Evaluation of An I226R and A137R deletion mutant from a genotype I/II recombinant African swine fever virus strain as a live attenuated vaccine in pigs

Evaluation of An I226R and A137R deletion mutant from a genotype I/II recombinant African swine fever virus strain as a live attenuated vaccine in pigs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of An I226R and A137R deletion mutant from a genotype I/II recombinant African swine fever virus strain as a live attenuated vaccine in pigs Han Zhang, Qixuan Li, Nannan Li, Tianying Hou, Hongliang Wang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9205759/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: The emergence of highly lethal genotype I/II recombinant African swine fever virus (ASFV) strains in China has rendered existing genotype II-based live attenuated vaccines ineffective, underscoring the urgent need for novel vaccine candidates. Although single-gene deletions of A137R or I226R have shown promise against genotype II strains, their efficacy and safety in the context of recombinant strains remain unexplored. Methods: Using homologous recombination, we constructed a single gene-deleted mutant (JX23-02ΔI226R) and a double gene-deleted mutant (JX23-02ΔI226RΔA137R) from the genotype I/II recombinant ASFV strain JX23-02. The replication kinetics, pathogenicity, immunogenicity, and protective efficacy of these mutants were evaluated in vitro and in pigs. Results: Both deletion mutants exhibited significantly reduced replication in porcine alveolar macrophages. Immunization with JX23-02ΔI226R resulted in 40% survival, but surviving pigs were fully protected against subsequent lethal challenge. In contrast, JX23-02ΔI226RΔA137R was completely attenuated: all immunized pigs survived challenge without any clinical signs and developed robust p54-specific antibody responses. Moreover, viral shedding and tissue viral loads were markedly lower in the double-deletion group than in the single-deletion group. Conclusions: The double gene-deleted mutant JX23-02ΔI226RΔA137R represents a safe and efficacious live attenuated vaccine candidate against genotype I/II recombinant ASFV strains, highlighting the superiority of multi-gene deletion strategies for ASF vaccine development. African swine fever virus Gene deletion I226R A137R Recombinant strain Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction African swine fever (ASF) is a severely lethal infectious disease affecting both domestic pigs and wild boars. Currently, apart from Vietnam, no safe, effective, and commercially available vaccine exists for ASF; therefore, disease control in other affected regions continues to rely primarily on culling, as ASF causes serious damage to the pig industry[ 1 ]. The disease was first reported in Kenya in 1921, and its causative agent is African swine fever virus (ASFV). ASFV is a large double-stranded DNA virus that is the only member of the genus Asfivirus, Asfarviridae family[ 2 ]. Based on the 3' terminal sequence of the B646L gene encoding the major capsid protein p72, ASFV was classified into 24 different genotypes. The virion is about 260 nm in diameter and has an icosahedral structure, and the viral genome is 170–193 kb and encodes more than 150 open reading frames[ 3 ]. Although the gene functions of many viral major proteins have been extensively studied[ 4 ], the same gene can still exhibit different virulence across different viral strains. For example, deletion of I267L in the ASFV-GS isolate results in loss of virulence in pigs, while this effect is not observed in the ASFV SY18 isolate[ 5 , 6 ]. This indicates that the gene functions in ASFV are highly intricate. Investigating the functions of the same gene in different viral strains will contribute to the development of live attenuated vaccines against ASF. Since the occurrence of ASF in China in 2018[ 7 ], it has been reported in several other Asian countries, such as South Korea[ 8 ] and Mongolia[ 9 ]. Due to the lack of safe and effective vaccines, ASF has spread widely and continues to evolve. In 2023, highly lethal genotype I and II recombinant strains were discovered in China, and attenuated live vaccine derived from genotype II cannot protect against the recombinant strains infection [ 10 ]. Therefore, it is necessary to develop vaccines targeting recombinant strains. The development of gene deleted live attenuated vaccines against ASF has made notable progress. Single gene-deletions, including I73R [ 11 ], I177L [ 12 ], MGF505-7R [ 13 ], A137R [ 14 ], and I226R [ 15 ], have been shown to provide complete protection against genotype II virulent challenge in experimental models. Nevertheless, the safety profiles of such monogenic mutants warrant careful evaluation. Similarly, multigene deletion strains face challenges such as reduced viral replication and suboptimal immunogenicity, which may limit their protective efficacy[ 16 – 18 ]. Recent advances in gene-deleted live attenuated vaccines have been comprehensively reviewed by Niu et al. (2025), who summarized the key virulence-related genes and discussed the prospects and challenges for ASFV vaccine development[ 19 ]. It is reported that immunization with A137R or I226R deleted attenuated strains provides complete protection against parental genotype II ASFV challenge with a favorable safety profile. Beyond I226R and A137R, other viral proteins have also been identified as critical modulators of host innate immunity. For instance, the H240R protein suppresses IL-1β production by targeting NF-κB signaling [ 20 ], and I267L inhibits RNA polymerase III-RIG-I-mediated innate immunity [ 6 ]. Mechanistically, I226R protein is known to impair antiviral responses, likely through multiple mechanisms including the suppression of NF-κB and IRF3 activation, to counteract innate immune responses [ 21 ]. As a virulence-associated gene, A137R inhibits cGAS-STING-mediated IFN-β production by promoting the autophagy-dependent lysosomal degradation of TBK1 [ 22 ]. Furthermore, studies indicate that A137R may also be implicated in mediating antibody-dependent enhancement (ADE) of infection [ 23 ]. Structural analysis reveals that A137R forms a dodecahedral cage, potentially contributing to ASFV icosahedral capsid assembly[ 24 ]. Collectively, these findings suggest that, apart from I177L , A137R and I226R represent promising targets for the development of live-attenuated ASF vaccines. However, most functional studies on these gene deletions have been conducted in the context of genotype II ASFV, and comparative analyses of their roles in other epidemiologically significant genotypes particularly in emerging genotype I/II recombinant strains is lacking, leaving their genotype-dependent effects on safety and immunogenicity unclear. Moreover, the protective efficacy and safety profile of a recombinant I226R or A137R deletion in a non-genotype II background remain unexplored. The global spread of ASF continues to threaten the swine industry, with sustained transmission in endemic regions and sporadic incursions into previously disease-free areas [ 25 ]. The development of safe and effective vaccines is therefore a global priority. Therefore, to address these gaps, this study was designed to generate and compare isogenic mutants derived from the genotype I/II recombinant strain JX23-02, featuring single deletions of I226R , as well as a double A137R / I226R deletion mutant. We aimed to systematically evaluate the impact of these deletions on viral replication, attenuation, safety, and protective efficacy within this novel recombinant background. This work not only provides critical insights for vaccine design against emerging recombinant strains but also enriches our understanding of the genotype-specific functions of A137R and I226R . 2. Material and methods 2.1 Cells and viruses Porcine alveolar macrophages (PAMs) and bone marrow-derived macrophages (BMDMs) were isolated from the bronchoalveolar lavage fluid and bone marrow aspirates of approximately 2-month-old pigs. The lavage fluids were supplemented with RPMI 1640 medium containing ethylenediaminetetraacetic acid (EDTA) and levofloxacin. After centrifugation, red blood cells were lysed using a red blood cell lysis buffer. The resultant cells were then washed three times with phosphate-buffered saline (PBS) containing levofloxacin and 2% fetal bovine serum (FBS), followed by resuspension in RPMI 1640 medium containing levofloxacin and 10% FBS. For the BMDMs, 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) was added to the resuspension. The cells were cultured for 7 days at 37°C with 5% CO 2 in the presence of GM-CSF before use. ASFV strain JX23-02 (GenBank accession: PP712069), isolated from pig spleen in 2023, is a genotype I/II recombinant variant and is preserved in our laboratory. 2.2 Plasmid design for traditional recombination The pMD18-T vector was used as the backbone. An EGFP expression cassette was inserted into the cloning site of pMD18-T. This cassette consisted of: a 1,258 bp left homology arm located upstream of the I226R open reading frame (ORF), the p72 promoter, the enhanced green fluorescent protein (EGFP) gene, an SV40 poly(A) termination signal, and a 1,114 bp right homology arm located downstream of the I226R ORF. To preserve the integrity of the I243L gene during deletion of I226R, a 22-bp sequence from the 3' end of the I226R gene was retained. Separately, an mCherry expression cassette was inserted into the cloning site of pMD18-T. This cassette comprised: a 1,061-bp left homology arm located upstream of the A137R ORF, the p72 promoter, the mCherry fluorescent protein gene, an SV40 poly(A) signal, and a 1,238 bp right homology arm located downstream of the A137R ORF. 2.3 Construction of gene-deletion JX23-02ΔI226R and JX23-02ΔI226RΔA137R Using the homologous recombination method, the I226R gene was replaced with EGFP gene to construct the I226R gene-deletion recombinant in JX23-02. Two micrograms of the recombinant plasmid, which contains the left and right homologous arms and the p72 promoter-operated EGFP gene, were transfected into BMDMs in a 12-well plate. After 6 hours, the parental strain was inoculated at a multiplicity of infection (MOI) of 1.0. Upon the emergence of fluorescent cells, a single one was picked under a microscope for isolation, followed by separating the gene-deletion strain from the parental strain using the method of limited dilution. PCR was performed to confirm the absence of the parental virus in the purified JX23-02ΔI226R strain. If the parental strain JX23-02 was present, an 819 bp band would be amplified using the following primers: forward: 5’-CCAATAGGCAACTTTCTTTTG − 3’; reverse: 5’-ACAGGATAACGATGCCCTTA-3’. Using the same method, a dual-gene deletion virus JX23-02ΔI226RΔA137R was constructed by transfecting a recombinant expression plasmid containing about the A137R gene left and right homologous arms and the p72 promoter-operated mCherry gene. After transfection, the purified JX23-02ΔI226R was inoculated at an MOI of 1.0. The recombinant virus was purified by picking individual fluorescent cells and employing limited dilution methods. Identification of the recombinant was performed by PCR, which amplified a 389 bp band using the following primers: forward: 5’-CAGTTCTTACCAAACTCGACC-3’; reverse: 5’-CATCTTGCCGATGAGATTTC-3’. 2.4 Viral growth curve assay To compare the growth curve of JX23-02, JX23-02ΔI226R, JX23-02ΔI226RΔA137R in Porcine alveolar macrophages (PAMs), each virus was inoculated at an MOI of 0.1 in triplicate of PAMs. After incubation for 1.5h, cells were washed with phosphate-buffered saline (PBS) and samples were collected at 24, 48, 72, 96, 120 h after infection, respectively. Subsequently, PAMs were used to detect the tissue culture infectious dose (TCID 50 ) of virus solution at different time points. The viral titer was detected by the tissue culture infectious dose (TCID 50 ) method and the direct immunofluorescence method. PAMs were resuscitated and then seeded into 96-well plates. Twelve hours later, the virus stocks at passages P2-P5 were repeatedly frozen and thawed, followed by 10-fold serial dilutions (from 10 1.0 to 10 8.0 ). Subsequently, 100 µl of the diluted virus solution per well was inoculated into the 96-well plates. The cells were continuously cultured for 96 hours under the conditions of 37°C and 5% CO₂. The cells were then fixed with 4% paraformaldehyde and placed at 4°C for 20 minutes. After washing the cells with phosphate-buffered saline (PBS), 0.1% Triton X-100 was added and the cells were treated for 10 minutes. The cells were washed again with PBS, and then the diluted FITC-conjugated p30 monoclonal antibody (at a dilution ratio of 1:500) was added. The cells were incubated at 37°C in the dark for 60 minutes. After that, the cells were washed three times with PBST (0.05% Tween 20), with each washing lasting for 3 minutes. The washing solution was discarded, and the number of fluorescent wells was observed and counted under a fluorescence microscope. Finally, the viral titer was calculated according to the Reed-Muench method. 2.5 Animal experiment To explore the pathogenicity of genotype I/II recombinant strains in pigs, 18 Landrace pigs were randomly divided into four groups. Group A (061, 023, 042, 001, 091), Group B (100, 004, 007, 006, 066) and Group C (275, 276, 277, 278, 279) were the infection groups, with 5 pigs in each group. Group D (901, 902, 903) was the control group, consisting of 3 pigs. Pigs in the first three groups were inoculated intramuscularly with 1 mL of ASFV SY18, 1 mL of HuB20 and 1 mL of JX23-02 respectively in the neck, all at a titer of 100 TCID 50 . Pigs in Group D were injected with 1 mL of PBS as a blank control group. The rectal temperatures were measured daily, and their feeding and mental status were observed. If the infected pigs showed severe clinical signs, they were humanely euthanized. The observation lasted for 28 days. To assess the effects of deleting the I226R gene and both the I226R and A137R genes on the virulence of the parental JX23-02 strain, 15 Landrace pigs were acquired from local farms for the experiment. The first group of 5 pigs (810,812,813,817,818) was intramuscularly inoculated with 10 5.0 TCID 50 of JX23-02ΔI226R, while another group of 5 pigs (081,082,083,084,085) was immunized with 10 5.0 TCID 50 of JX23-02ΔI226RΔA137R. The third group of 5 pigs (292,293,294,261,262) served as the challenge control. After inoculation, the clinical signs of the animals were monitored on a daily basis. For the surviving pigs during the immunity observation period, a challenge infection was performed via intramuscular injection of the parental strain ASFV JX23-02 at a dose of 100 TCID₅₀. Rectal temperature was taken daily, and oral and rectal swabs as well as blood samples were collected for the detection of viral nucleic acid and antibody levels in 0,3,7,10,14,17,21 days after inoculation and in 0,3,7,10,14,17,21,24,28 days after challenge. A part of the blood samples was preserved in EDTA blood collection tubes for viral load detection, and the other part was used for serum separation to detect antibodies. During the observation period, animals with severe clinical signs were humanely euthanized and collected the tissue samples including the heart, liver, spleen, lung, kidney, stomach, lymph nodes (submandibular and inguinal), thymus, tonsils, jejunum, ileum, colon, muscle, bone marrow, and quantitative polymerase chain reaction (qPCR) was carried out on tissue samples. The animal experiments were conducted in biosafety level 3 (BSL-3) laboratories. 2.6 Quantification of the ASFV load The quantification of p72 gene copies in blood, oral swabs, anal swabs, and tissues was conducted using qPCR protocols recommended by the World Organization for Animal Health (WOAH) ( https://www.woah.org/en/home/ ). Viral copy numbers were determined by qPCR using a standard curve based on the p72 gene, as previously described[ 26 ]. Tissue samples were cut into approximately 1 cm³ pieces and placed into sterile tubes containing steel beads. Subsequently, 1 mL of PBS was added, and the tissues were homogenized at 60 oscillations/s for 30 s using a tissue homogenizer; this cycle was repeated three times with 10-s intervals between cycles. The homogenates were then centrifuged at 12,000×g for 5 min at 4°C. The supernatants were collected, transferred to new sterile microcentrifuge tubes, and labeled for subsequent use. Briefly, DNA was extracted from each sample using an animal virus DNA extraction kit. Quantitative PCR (qPCR) was performed using the Premix Ex Taq (Probe qPCR) kit. The primers and probes used are listed in the additional table 1. The copy numbers of ASFV genomic DNA in samples were calculated based on a standard curve established using a standard plasmid containing the ASFV B646L gene. The standard curve for the ASFV p72 gene in the quantitative PCR was fitted to the equation Y= − 3.245X + 38.439, where Y represents the Ct value and X represents the copy number of the p72 gene. 2.7 Antibody detection The indirect ELISA method, developed in our laboratory, was used to detect the level of ASFV p54 specific antibodies in serum. The ELISA plate was coated with p54 antibody and incubated overnight at 4°C. After blocking, a 50-fold diluted sample serum was added and incubated at 37°C for 30 minutes. The plate was washed five times with PBS, then incubated with Horseradish peroxidase (HRP)-labeled sheep anti-pig IgG at 37°C for 30 minutes. Following the wash, 80 µl of chromogenic solution 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to each well, incubated at room temperature in the dark for 10 minutes, then the reaction was stopped with 2 M sulfuric acid, and read within 3 minutes. Each serum sample was tested three times, and their absorbance values were measured at 450 nm. 2.8 Statistical analysis GraphPad Prism 9.5 software was used for graphing and statistical analysis. The t-test was employed for significance analysis. "ns" indicates no significant difference, "*" represents a significant difference ( P < 0.05), "**" indicates an extremely significant difference ( P < 0.01), and "***" represents an extremely significant difference ( P < 0.001). 3. Results 3.1 The pathogenicity of JX23-02. Georgia07-like genotype II strain SY18 is highly lethal strain and HuB20 is naturally attenuated genotype II strain.To determine genotype I/ II recombinant strain virulence with genotype II, pigs were infected with the strains of JX23-02, HuB20 and SY18 at a dose of 100 TCID 50 respectively and measured the rectal temperature daily. Pigs in the control group remained clinically normal throughout the experiment. These three strains demonstrated a 100% mortality rate (Fig. 1 A). Among them, the pigs infected with the JX23-02 strain developed fever as early as 2 days post-infection (dpi), and the fever lasted for 1 to 3 days, with the earliest acute death occurring at 5 dpi. The pigs infected with the SY18 strain developed fever at 3 dpi, the fever persisted until around 5 dpi (Fig. 1 B), and showed signs of being on the verge of death as early as 7 dpi. The pigs infected with the HuB20 strain developed fever at 11 dpi, which lasted for about 10 days, and started to die as early as 22 dpi. 3.2 Construction and growth characteristics of JX23-02ΔI226R and JX23-02ΔI226RΔA137R deletion strains. The construction strategy of the ASFV gene-deleted strain is illustrated in Fig. 2 A, where the I226R gene was replaced with the p72-EGFP cassette via homologous recombination. Since the terminal ends of the I226R and I243L open reading frames (ORFs) share 4 base pairs, when deleting the I226R ORF, it is necessary to avoid disrupting the I243L ORF. Therefore, 673 base pairs of the I226R were deleted to protect the integrity of the I243L ORF. After transfection and infection of BMDMs, the fluorescent cell lesions were collected. After multiple rounds of limiting dilution, virus expressing green fluorescence was purified and named JX23-02ΔI226R(Fig. 2 C). Routine polymerase chain reaction (PCR) was performed on the purified virus using differential primers, and no band of 819 base pairs in size was generated, indicating that the purification was successful (Fig. 2 E). The A137R gene was completely deleted based on the JX23-02ΔI226R strain, and the A137R gene locus was replaced with the p72-mCherry cassette. After purification, a recombinant virus emitting both red and green fluorescence was obtained and designated as JX23-02ΔI226RΔA137R (Fig. 2 D). Through PCR identification, the purified JX23-02ΔI226RΔA137R strain was obtained(Fig. 2 F, 2 G). To elucidate the effects of I226R and A137R deletion on ASFV JX23-02 replication, we evaluated the replication kinetics of JX23-02ΔI226R and ASFV JX23-02ΔI226RΔA137R in BMDMs. JX23-02ΔI226R or ASFV JX23-02ΔI226RΔA137R was infected at an MOI of 0.01, and the virus yields were detected at 0, 24, 48, 72, 96, and 120 hours post-infection (hpi), respectively. The results showed that compared with the parental strain, the viral titers of the recombinant strains JX23-02ΔI226R and JX23-02ΔI226RΔA137R were significantly reduced at 24 hpi, 48 hpi, and 72 hpi ( P < 0.01). At 96 hpi and 120 hpi, the viral titers of the parental strain were significantly higher ( P < 0.05) than those of the double gene-deleted strain JX23-02ΔI226RΔA137R, while there was no significant difference in the titers between the single gene-deleted strain JX23-02ΔI226R and the parental strain at 120 hpi ( P > 0.05). In addition, the titers of the double gene-deleted strain JX23-02ΔI226RΔA137R were slightly lower than those of the single gene-deleted strain JX23-02ΔI226R at 72 hpi and 96 hpi, but there was no statistically significant difference between them ( P > 0.05). Overall, the deletion of the I226R and A137R genes had a stage-specific effect on the in vitro replication ability of the ASFV JX23-02 strain, especially during the middle stage of infection (24–72 hpi). The specific results are shown in Fig. 2 B. 3.3 JX23-02ΔI226R exhibits residual virulence but provides protection in surviving pigs. JX23-02ΔI226R attenuated part of the virulence of the parental virus strain and caused the death of some pigs. However, the deletion strain also provided complete protection to the surviving pigs against the challenge of the parental virus strain. Five pigs were immunized intramuscularly with a dose of 10 5.0 TCID 50 of JX23-02ΔI226R. Three pigs (810, 817, 818) died on days 11, 12, and 16 post-immunization, respectively. The deceased pigs exhibited typical clinical signs of ASF, including fever, depression, lethargy, and anorexia, with peak body temperatures reaching 41.5°C [ 27 ]. The remaining two pigs showed normal performance within the 21-day observation period, resulting in a survival rate of 40% (Fig. 3 A). The pigs started to develop fever on the third day after immunization, with the fever reaching its peak from the 7–10 dpi and lasting for 5–13 days. Two pigs exhibited pyrexia, with individual peak temperatures reaching 40.9°C and 40.8°C (Fig. 3 B). The febrile response was accompanied by anorexia and reduced mobility, and both animals recovered fully within 3–4 days after symptom onset. 21 days after immunization, the pigs were challenged intramuscularly with a dose of 100 TCID 50 . The body temperatures of the two surviving pigs immunized with JX23-02ΔI226R increased transiently on 7 to 9 post-challenge (dpc) and then returned to normal(Fig. 3 C). They survived with normal physiological performance within the 28-day observation period. In the control group, the body temperatures of the pigs increased sharply on the 3rd day after challenge, and died at 5 dpc. All pigs in the control group died within 8 dpc (Fig. 3 A). 3.4 JX23-02ΔI226RΔA137R can be completely attenuated and provides complete protective efficacy against the parental virus strain JX23-02ΔI226RΔA137R is completely attenuated relative to the parental virus strain and can provide full protection to immunized pigs. Five pigs were immunized intramuscularly with a dose of 10 5.0 TCID 50 of JX23-02ΔI226RΔA137R. All pigs survived during the immunization observation period and also after the challenge with the parental virus strain. The pigs started to have a slight fever on the 5th day after immunization, which lasted for 1–3 days. There was some fluctuation in body temperature during the observation period, but the maximum body temperature did not exceed 40.1°C. 21 days after immunization, the five pigs were challenged with 100 TCID 50 of the parental virus strain JX23-02. All pigs survived, with their body temperatures remaining basically normal and no obvious fever symptoms shown. 3.5 Occasional shedding occurs through the oral or anal route after JX23-02ΔI226R and JX23-02ΔI226RΔA137R inoculation and ASFV JX23-02 challenge. During the observation period after immunization and challenge, oral and anal swabs were collected from pigs, DNA was extracted, and viral load was detected by qPCR. The detection results of oral and anal swabs after immunization are shown in Fig. 4 . In the pigs immunized with JX23-02ΔI226R, the oral and anal swabs showed positive results in 4 pigs during the early stage (within 7 to 14 days) after vaccination, and viremia was detected in all these pigs. In contrast, in the pigs immunized with JX23-02ΔI226RΔA137R, all swab samples tested negative. The amplification results of the p72 gene on the oral and anal swabs after challenge infection showed that in the control group pigs, viral shedding was detected starting from 3 days after challenge, and the copy number of viral DNA was relatively high. In the oral and anal swabs of the JX23-02ΔI226R immunized group, viral shedding was detected in 1 to 2 pigs. In the JX23-02ΔI226RΔA137R immunized group, viral DNA was only detected in the anal swab of one pig, showing a weakly positive result. This suggests that JX23-02ΔI226RΔA137R may have an improved safety profile compared to JX23-02ΔI226R. 3.6 JX23-02ΔI226RΔA137R induces lower viremia and tissue viral load compared to JX23-02ΔI226R We performed qPCR on the whole blood of pigs collected after immunization and challenge to detect the viral genome content. Autopsies were carried out on each dead pig. All organs of the dead pigs in the control group and the JX23-02ΔI226R immunization group showed typical gross pathological changes of African swine fever, such as splenomegaly and visceral hemorrhage (Fig. 5 A.5C). Aside from a few petechial hemorrhages observed in the lungs, no obvious pathological changes were observed in the pigs of the JX23-02ΔI226RΔA137R immunization group. (Fig. 5 B). Organ tissue samples were collected for quantitative detection of the viral genome. In the blood samples after immunization, viral DNA was detected at high levels in the blood of control group pigs after challenge, and the viral DNA copy number in various tissues was also high. The viral content in the blood of pigs immunized with JX23-02ΔI226R was significantly higher than that in the JX23-02ΔI226RΔA137R group. The viral genome in the blood was undetectable (negative) by qPCR 28 days after challenge. In the JX23-02ΔI226R group, the viral load in the tissues of the dead pigs after immunization was high, and qPCR was positive in all types of tissue samples. In the surviving pigs, the virus was detected in the colon, jejunum, and bone marrow, indicating that the virus in the tissues and organs could not be completely cleared. In the JX23-02ΔI226RΔA137R group, viremia disappeared 21 days after challenge, and the blood qPCR test was negative. In the detection of the viral genome in tissue samples, the kidney, inguinal lymph node, thymus, and stomach tissues tested negative, while the viral genome was detected in other tissues, but only a limited number of samples tested positive. 3.7 JX23-02ΔI226R and JX23-02ΔI226RΔA137R induced strong immune response The kinetics of p54-specific antibody production were consistent with those typically observed for attenuated ASFV vaccines. As illustrated in Fig. 6 B, antibody levels were detectable in all experimental groups on day 7 post-vaccination, peaking approximately between days 17 and 21 post-vaccination. Following challenge, the p54 antibody levels in the experimental groups slightly increased and then declined moderately over time, but remained at a high positive level throughout the observation period. In contrast, p54 antibodies were only detected at a low level in the control group after challenge. 4 Discussion In the present study, we successfully constructed a single gene-deleted strain (JX23-02ΔI226R) and a double gene-deleted strain (JX23-02ΔI226RΔA137R) derived from the genotype I/II recombinant ASFV strain JX23-02. The emergence of genotype I/II recombinant ASFV strains poses new challenges for vaccine development, as existing genotype II-based vaccines have shown limited protection against these variants [ 10 ]. Our successful attenuation of the recombinant strain JX23-02 through gene deletion demonstrates that targeted modification of circulating field strains is a feasible strategy to address this challenge. Subsequently, we systematically evaluated their immunogenicity, protective efficacy, and safety as potential vaccine candidates. A key finding of this study is that the deletion of I226R and A137R genes significantly impaired the in vitro replication capacity of ASFV, particularly during the middle stage of infection (24–72 h), as evidenced by a marked reduction in viral titer. This observation provides critical theoretical insights into the development of attenuated ASFV vaccines, highlighting the potential of I226R and A137R as key virulence-related targets for viral attenuation. Animal experiments revealed distinct levels of immune protection among the constructed strains. Specifically, JX23-02ΔI226R retained residual virulence, which led to the death of some immunized pigs; however, it still induced effective protective immune responses against the parental strain, indicating its potential as a vaccine candidate with room for further optimization. In contrast, JX23-02ΔI226RΔA137R exhibited superior safety profiles: all immunized pigs survived the challenge without manifesting obvious clinical signs. This result demonstrates that the additional deletion of the A137R gene further attenuated viral virulence without compromising immunogenicity, confirming the synergistic effect of dual gene deletion on ASFV attenuation. Furthermore, qPCR analysis showed that pigs immunized with JX23-02ΔI226RΔA137R had significantly shorter duration of viremia and lower viral load compared to those in the JX23-02ΔI226R group. These data further validate the advantages of JX23-02ΔI226RΔA137R in suppressing viral replication and reducing viremia persistence, thereby providing robust support for its potential as a promising vaccine candidate. The enhanced safety profile of JX23-02ΔI226RΔA137R may be attributed to the cumulative effect of deleting two immunomodulatory genes. I226R suppresses NF-κB and IRF3 activation [ 21 ], while A137R inhibits cGAS-STING-mediated IFN-β production by promoting autophagy-dependent degradation of TBK1 [ 22 ]. Simultaneous deletion of both genes likely leads to more robust innate immune responses against the virus, thereby limiting its replication and dissemination in vivo, which explains the reduced viremia and lower tissue viral load observed in this group. Collectively, based on reduced viremia, lower tissue viral load, and absence of mortality, JX23-02ΔI226RΔA137R outperformed JX23-02ΔI226R in terms of safety, immunogenicity, and viral replication control, offering valuable experimental basis and theoretical support for the development of ASFV vaccines. Our findings exhibit both similarities and discrepancies with previous reports on ASFV gene-deleted vaccines. Zhang et al.[ 28 ] demonstrated that the SY18ΔI226R strain did not induce obvious clinical signs after vaccination and conferred complete protection against lethal challenge with a virulent strain. While JX23-02ΔI226R in the present study also provided partial protective efficacy, its higher residual virulence resulted in partial mortality among immunized animals. This discrepancy is likely attributed to the inherent genetic background differences and distinct pathogenic mechanisms between the parental strains (SY18 and JX23-02), emphasizing the importance of tailoring attenuation strategies to specific ASFV genotypes or recombinant strains. On the other hand, Zhao et al. reported that the genotype II attenuated live vaccine HLJ/18-7GD failed to protect against genotype I/II recombinant ASFV strains, suggesting that genomic recombination of ASFV may lead to the emergence of novel epitopes or immune escape mechanisms, thereby compromising the protective efficacy of existing vaccines [ 29 ]. The successful development of JX23-02ΔI226RΔA137R in this study addresses this challenge by proposing a multi-gene deletion strategy, which achieves enhanced attenuation while preserving critical immunogenic determinants. Recently, Peng et al. demonstrated that deletion of CD2v and A137R from another genotype II strain also resulted in complete attenuation and protection [ 30 ]. While their double-deletion strategy targeted different genes (CD2v and A137R), our results with JX23-02ΔI226RΔA137R similarly confirm that A137R is a key target for attenuation across different genetic backgrounds, and suggest that multiple gene combinations can achieve optimal safety and efficacy. In conclusion, we successfully constructed two gene-deleted strains (JX23-02ΔI226R and JX23-02ΔI226RΔA137R) based on the genotype I/II recombinant ASFV strain JX23-02. Deletion of I226R and A137R significantly attenuated viral replication capacity. Among the candidates, JX23-02ΔI226RΔA137R exhibited excellent performance in safety, immunogenicity, and protective efficacy, whereas JX23-02ΔI226R induced partial protective immunity but retained undesirable residual virulence. These results highlight the feasibility of multi-gene deletion as a promising approach for the development of safe and effective ASFV vaccines, especially against emerging recombinant strains. Declarations Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee of Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences (protocol number: IACUC of AMMS-11-2024-67) and were conducted in accordance with relevant guidelines and regulations. Availability of data and materials The ASFV strain JX23-02 genome sequence is available in GenBank under accession number PP712069. Competing interests The authors declare that they have no competing interests. Funding This work is financially supported by the Foundation of State Key Laboratory of Pathogen and Biosecurity (grant No. SKLPBS2421) and the National Key Research and Development Program of China (Grant No. 2021YFD1800100). The funding body had no role in the design of the study, data collection, analysis, interpretation, or writing of the manuscript. Authors' contributions Conceptualization: HZ, RH, SZ, FM; methodology: HZ, QL, NL, TH; validation: RH, FM; data collection: HZ, QL; writing—original draft preparation: HZ; writing—review and editing: QL, NL, TH, HW, RH, SZ, FM; supervision: RH, SZ, FM. All authors read and approved the final manuscript. References van den Born E., Olasz F., Mészáros I., Göltl E., Oláh B., Joshi J., van Kilsdonk E., Segers R., Zádori Z., African swine fever virus vaccine strain Asfv-G-∆I177l reverts to virulence and negatively affects reproductive performance, NPJ Vaccines. (2025) 10:46. Alonso C., Borca M., Dixon L., Revilla Y., Rodriguez F., Escribano J.M., ICTV Virus Taxonomy Profile: Asfarviridae, J. Gen. Virol. (2018) 99:613-614. Wang N., Zhao D., Wang J., Zhang Y., Wang M., Gao Y., Li F., Wang J., Bu Z., Rao Z., Wang X., Architecture of African swine fever virus and implications for viral assembly, Science. (2019) 366:640-644. Wang G., Xie M., Wu W., Chen Z., Structures and Functional Diversities of ASFV Proteins, Viruses. (2021) 13. Zhang Y., Ke J., Zhang J., Yue H., Chen T., Li Q., Zhou X., Qi Y., Zhu R., Wang S., Miao F., Zhang S., Li N., Mi L., Yang J., Yang J., Han X., Wang L., Li Y., Hu R., I267L Is Neither the Virulence- Nor the Replication-Related Gene of African Swine Fever Virus and Its Deletant Is an Ideal Fluorescent-Tagged Virulence Strain, Viruses. (2021) 14. Ran Y., Li D., Xiong M.G., Liu H.N., Feng T., Shi Z.W., Li Y.H., Wu H.N., Wang S.Y., Zheng H.X., Wang Y.Y., African swine fever virus I267L acts as an important virulence factor by inhibiting RNA polymerase III-RIG-I-mediated innate immunity, PLoS Pathog. (2022) 18:e1010270. Zhou X., Li N., Luo Y., Liu Y., Miao F., Chen T., Zhang S., Cao P., Li X., Tian K., Qiu H.-J., Hu R., Emergence of African Swine Fever in China, 2018, Transboundary and Emerging Diseases. (2018) 65:1482-1484. Kim S.-H., Kim J., Son K., Choi Y., Jeong H.-S., Kim Y.-K., Park J.-E., Hong Y.-J., Lee S.-I., Wang S.-J., Lee H.-S., Kim W.-M., Jheong W.-H., Wild boar harbouring African swine fever virus in the demilitarized zone in South Korea, 2019, Emerging Microbes & Infections. (2020) 9:628-630. Ankhanbaatar U., Sainnokhoi T., Khanui B., Ulziibat G., Jargalsaikhan T., Purevtseren D., Settypalli T.B.K., Flannery J., Dundon W.G., Basan G., Batten C., Cattoli G., Lamien C.E., African swine fever virus genotype II in Mongolia, 2019, Transbound Emerg Dis. (2021) 68:2787-2794. Zhao D., Sun E., Huang L., Ding L., Zhu Y., Zhang J., Shen D., Zhang X., Zhang Z., Ren T., Wang W., Li F., He X., Bu Z., Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs, Nat Commun. (2023) 14. Liu Y., Shen Z., Xie Z., Song Y., Li Y., Liang R., Gong L., Di D., Liu J., Liu J., Chen Z., Yu W., Lv L., Zhong Q., Liao X., Tian C., Wang R., Song Q., Wang H., Peng G., Chen H., African swine fever virus I73R is a critical virulence-related gene: A potential target for attenuation, Proceedings of the National Academy of Sciences. (2023) 120. Borca M.V., Ramirez-Medina E., Silva E., Vuono E., Rai A., Pruitt S., Espinoza N., Velazquez-Salinas L., Gay C.G., Gladue D.P., ASFV-G-∆I177L as an Effective Oral Nasal Vaccine against the Eurasia Strain of Africa Swine Fever, Viruses. (2021) 13. Li J., Song J., Kang L., Huang L., Zhou S., Hu L., Zheng J., Li C., Zhang X., He X., Zhao D., Bu Z., Weng C., pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1β and type I IFN production, PLoS Pathog. (2021) 17:e1009733. Gladue D.P., Ramirez-Medina E., Vuono E., Silva E., Rai A., Pruitt S., Espinoza N., Velazquez-Salinas L., Borca M.V., Deletion of the A137R Gene from the Pandemic Strain of African Swine Fever Virus Attenuates the Strain and Offers Protection against the Virulent Pandemic Virus, J. Virol. (2021) 95:e0113921. Zhang Y., Ke J., Zhang J., Yang J., Yue H., Zhou X., Qi Y., Zhu R., Miao F., Li Q., Zhang F., Wang Y., Han X., Mi L., Yang J., Zhang S., Chen T., Hu R., African Swine Fever Virus Bearing an I226R Gene Deletion Elicits Robust Immunity in Pigs to African Swine Fever, J. Virol. (2021) 95. Gladue D.P., O'Donnell V., Ramirez-Medina E., Rai A., Pruitt S., Vuono E.A., Silva E., Velazquez-Salinas L., Borca M.V., Deletion of CD2-Like (CD2v) and C-Type Lectin-Like (EP153R) Genes from African Swine Fever Virus Georgia-∆9GL Abrogates Its Effectiveness as an Experimental Vaccine, Viruses. (2020) 12. Ramirez-Medina E., Vuono E., O'Donnell V., Holinka L.G., Silva E., Rai A., Pruitt S., Carrillo C., Gladue D.P., Borca M.V., Differential Effect of the Deletion of African Swine Fever Virus Virulence-Associated Genes in the Induction of Attenuation of the Highly Virulent Georgia Strain, Viruses. (2019) 11. O'Donnell V., Holinka L.G., Sanford B., Krug P.W., Carlson J., Pacheco J.M., Reese B., Risatti G.R., Gladue D.P., Borca M.V., African swine fever virus Georgia isolate harboring deletions of 9GL and MGF360/505 genes is highly attenuated in swine but does not confer protection against parental virus challenge, Virus Res. (2016) 221:8-14. Niu X., Shen D., Bu Z., Dixon L.K., Zhao D., Hope and hurdles: unlocking the potential of modified live virus vaccines for African swine fever, Emerg Microbes Infect. (2025) 14:2572692. Zhou P., Dai J., Zhang K., Wang T., Li L.F., Luo Y., Sun Y., Qiu H.J., Li S., The H240R Protein of African Swine Fever Virus Inhibits Interleukin 1β Production by Inhibiting NEMO Expression and NLRP3 Oligomerization, J Virol. (2022) 96:e0095422. Hong J., Chi X., Yuan X., Wen F., Rai K.R., Wu L., Song Z., Wang S., Guo G., Chen J.L., I226R Protein of African Swine Fever Virus Is a Suppressor of Innate Antiviral Responses, Viruses. (2022) 14. Sun M., Yu S., Ge H., Wang T., Li Y., Zhou P., Pan L., Han Y., Yang Y., Sun Y., Li S., Li L.F., Qiu H.J., The A137R Protein of African Swine Fever Virus Inhibits Type I Interferon Production via the Autophagy-Mediated Lysosomal Degradation of TBK1, J Virol. (2022) 96:e0195721. Yang X., Sun E., Zhai H., Wang T., Wang S., Gao Y., Hou Q., Guan X., Li S., Li L.F., Wu H., Luo Y., Li S., Sun Y., Zhao D., Li Y., Qiu H.J., The antibodies against the A137R protein drive antibody-dependent enhancement of African swine fever virus infection in porcine alveolar macrophages, Emerg Microbes Infect. (2024) 13:2377599. Li C., Jia M., Hao T., Peng Q., Peng R., Chai Y., Shi Y., Song H., Gao G.F., African swine fever virus A137R assembles into a dodecahedron cage, J. Virol. (2024) 98:e0153623. Dixon L.K., Stahl K., Jori F., Vial L., Pfeiffer D.U., African Swine Fever Epidemiology and Control, Annu Rev Anim Biosci. (2020) 8:221-246. Zuo X., Peng G., Xia Y., Xu L., Zhao Q., Zhu Y., Wang C., Liu Y., Zhao J., Wang H., Zou X., A quadruple fluorescence quantitative PCR method for the identification of wild strains of african swine fever and gene-deficient strains, Virol J. (2023) 20:150. Zuo X., Peng G., Zhao J., Zhao Q., Zhu Y., Xu Y., Xu L., Li F., Xia Y., Liu Y., Wang C., Wang Z., Wang H., Zou X., Infection of domestic pigs with a genotype II potent strain of ASFV causes cytokine storm and lymphocyte mass reduction, Front Immunol. (2024) 15:1361531. Zhang Y., Ke J., Zhang J., Yang J., Yue H., Zhou X., Qi Y., Zhu R., Miao F., Li Q., Zhang F., Wang Y., Han X., Mi L., Yang J., Zhang S., Chen T., Hu R., African Swine Fever Virus Bearing an I226R Gene Deletion Elicits Robust Immunity in Pigs to African Swine Fever, J. Virol. (2021) 95:e0119921. Zhao D., Sun E., Huang L., Ding L., Zhu Y., Zhang J., Shen D., Zhang X., Zhang Z., Ren T., Wang W., Li F., He X., Bu Z., Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs, Nat Commun. (2023) 14:3096. Peng G., Zhao X., Zou X., Zhang H., Zhao J., Zuo X., Tan S., Wu R., Guan X., Li S., Xu Y., Xia Y., Xu X., Xu L., Zhu Y., Liu J., Liu Y., Gao G.F., An attenuated African swine fever virus with deletions of the CD2v and A137R genes offers complete protection against homologous challenge in pigs, J Virol. (2025) 99:e0026225. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9205759","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618640334,"identity":"f7164c37-ee15-4d9d-933f-4a0cf0bf131a","order_by":0,"name":"Han Zhang","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Zhang","suffix":""},{"id":618640335,"identity":"f9c24e77-e962-46a8-a466-456eb6da4f17","order_by":1,"name":"Qixuan Li","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qixuan","middleName":"","lastName":"Li","suffix":""},{"id":618640336,"identity":"ba9a362c-b136-44a1-9155-02f19ef80c5f","order_by":2,"name":"Nannan Li","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nannan","middleName":"","lastName":"Li","suffix":""},{"id":618640337,"identity":"24bcf85c-1f39-4328-b880-ebc11d1f6151","order_by":3,"name":"Tianying Hou","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tianying","middleName":"","lastName":"Hou","suffix":""},{"id":618640338,"identity":"9db9bf35-f78f-4cb5-9809-71bd4d846e4c","order_by":4,"name":"Hongliang Wang","email":"","orcid":"","institution":"Institute of Animal Husbandry and Veterinary Medicine, Heilongjiang Academy of Agricultural Reclamation Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hongliang","middleName":"","lastName":"Wang","suffix":""},{"id":618640339,"identity":"cdb6c8ab-66b9-4bcc-b800-8cfc3ddbf53d","order_by":5,"name":"Rongliang Hu","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Rongliang","middleName":"","lastName":"Hu","suffix":""},{"id":618640340,"identity":"6ce4b83a-36d3-423e-b10f-5e764ced2266","order_by":6,"name":"Shoufeng Zhang","email":"","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shoufeng","middleName":"","lastName":"Zhang","suffix":""},{"id":618640341,"identity":"6cbc1972-abce-40e8-83a9-d36d04642ec2","order_by":7,"name":"Faming Miao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBAC9gYGhgNAmoeBgfkYWISNnYAWngNwLWxpDAwJQIqZCC0wphlYCwNBLeynEw8X/Nomw9/e8+3Bxx/b5PmYGRg/fMzBo4Und8PhmX23eSTOnN1uOCPhtmEbMwOz5MxtuLXYMwC18Pbc5mG4kbtNmifhNiNQCxszLx4tPPxvIVrkb+Q8A2mxJ6xFAmgLz4/bPAY3cthAWhKJ0AKypeE2j+GZY2aSM9JuJ7cxMzbj9QsPf+7mzzx/btvLHW9+JvHB5rbt/Pbmgx8+4tECBoxtqNwGAupB4A8RakbBKBgFo2DkAgDesVG5srClHgAAAABJRU5ErkJggg==","orcid":"","institution":"Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Faming","middleName":"","lastName":"Miao","suffix":""}],"badges":[],"createdAt":"2026-03-24 02:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9205759/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9205759/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106660833,"identity":"74360379-2639-4175-aa1e-6a4e82916729","added_by":"auto","created_at":"2026-04-11 06:05:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8563134,"visible":true,"origin":"","legend":"\u003cp\u003eHigh pathogenicity of JX23-02 in pigs. (A) Survival curves post-challenge with JX23-02, SY18, or HuB20. (B) Body temperature profiles post-challenge with the indicated strains.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9205759/v1/9d02e2ef461e8d86aff1dc69.png"},{"id":106660834,"identity":"b12b69fb-d596-4195-804b-1e76368c0f16","added_by":"auto","created_at":"2026-04-11 06:05:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63420835,"visible":true,"origin":"","legend":"\u003cp\u003eThe construction of gene-deleted ASFV JX23-02ΔI226R and JX23-02ΔI226RΔA137R (A)Schematic representation of JX23-02ΔI226R and JX23-02ΔI226RΔA137R construction. (B) In vitro replication kinetics of gene-deleted ASFV JX23-02ΔI226R and JX23-02ΔI226RΔA137R compared to ASFV JX23-02. (*, \u003cem\u003eP\u003c/em\u003e＜0.05; **, \u003cem\u003eP\u003c/em\u003e＜0.01;***,\u003cem\u003eP\u003c/em\u003e＜0.001) (C) The ASFV JX23-02ΔI226R infected BMDMs are indicated by EGFP reporter fluorescence. (D) The ASFV JX23-02ΔI226RΔA137R infected BMDMs are indicated by EGFP and mCherry reporter fluorescence. (E) The ASFV JX23-02ΔI226R genome is amplified using specific primers targeting \u003cem\u003eI226R\u003c/em\u003e. The ASFV JX23-02 genome is used as a positive control. Lane 1–2: \u003cem\u003eI226R\u003c/em\u003e gene amplification from purified JX23-02ΔI226R samples, Lane 3: negative control, Lane 4: \u003cem\u003eI226R\u003c/em\u003e gene in ASFV JX23-02. (F) The ASFV JX23-02ΔI226RΔA137R genome is amplified using specific primers targeting \u003cem\u003eA137R\u003c/em\u003e. Lane 1: \u003cem\u003eA137R\u003c/em\u003e gene amplification from purified JX23-02ΔI226RΔA137R samples, Lane 2: negative control, Lane 3: \u003cem\u003eA137R\u003c/em\u003egene in ASFV JX23-02. (G) The ASFV JX23-02ΔI226RΔA137R genome is amplified using specific primers targeting \u003cem\u003eI226R\u003c/em\u003e. Lane 1: \u003cem\u003eI226R\u003c/em\u003e gene amplification from purified JX23-02ΔI226RΔA137R samples, Lane 2: negative control, Lane 3: \u003cem\u003eI226R\u003c/em\u003e gene in ASFV JX23-02.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-9205759/v1/323d75f1a7056a4608ad1529.png"},{"id":107479611,"identity":"f0e22582-9ce3-4b39-9b44-86f4903dfede","added_by":"auto","created_at":"2026-04-22 01:29:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4810683,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival and body temperature responses to immunization and challenge. (A) Survival curves post-immunization and post-challenge. (B) Body temperature profiles post-immunization. (C) Body temperature profiles post-challenge.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-9205759/v1/7cc8fbaa18601c28f6910fe1.png"},{"id":106660837,"identity":"7cd56a54-4bdd-4217-9e08-af4d429bd134","added_by":"auto","created_at":"2026-04-11 06:05:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1651397,"visible":true,"origin":"","legend":"\u003cp\u003eViral genomic DNA detection in blood, oral swabs, and anal swabs from the three groups of pigs. (A, B) Viral genome copies in blood post-immunization and post-challenge, respectively. (C, D) Viral genome copies in anal and oral swabs post-immunization. (E, F) Viral genome copies in anal and oral swabs post-challenge.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-9205759/v1/858b4385707f1ab7359105e2.png"},{"id":106959077,"identity":"e0a3be24-3707-4c9b-a9f4-964e447b53dd","added_by":"auto","created_at":"2026-04-15 08:45:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eAnatomical characteristics of porcine tissues and organs. heart (a), liver (b), lung (c), kidney (d), inguinal lymph node (e), submandibular lymph node (f), thymus (g), and spleen (h).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9205759/v1/49738469e2c96dcf7eb7e543.png"},{"id":106660839,"identity":"1679d31a-6c7e-4987-bea4-c7a106696739","added_by":"auto","created_at":"2026-04-11 06:05:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1386817,"visible":true,"origin":"","legend":"\u003cp\u003eASFV genomic copy numbers in tissues and serum antibody detection results. (A) ASFV genomic copy numbers in tissues collected from pigs at necropsy.(B) Antibody to p54 response curves of different groups of pigs after inoculation and challenge.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-9205759/v1/fec5c75fb3893f9b3d343fb9.png"},{"id":109219744,"identity":"ec4e3640-c6f4-4a83-8167-4bf8c3dbb45d","added_by":"auto","created_at":"2026-05-13 20:02:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":72313713,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9205759/v1/05d4fbf8-d77d-4591-9515-12d242a9fb58.pdf"},{"id":106727645,"identity":"aceb0cbb-3ca7-4d69-85ed-47f5fb35d4c4","added_by":"auto","created_at":"2026-04-12 18:39:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14446,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9205759/v1/8f3feb3fa9d327bccacf7534.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of An I226R and A137R deletion mutant from a genotype I/II recombinant African swine fever virus strain as a live attenuated vaccine in pigs","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAfrican swine fever (ASF) is a severely lethal infectious disease affecting both domestic pigs and wild boars. Currently, apart from Vietnam, no safe, effective, and commercially available vaccine exists for ASF; therefore, disease control in other affected regions continues to rely primarily on culling, as ASF causes serious damage to the pig industry[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe disease was first reported in Kenya in 1921, and its causative agent is African swine fever virus (ASFV). ASFV is a large double-stranded DNA virus that is the only member of the genus \u003cem\u003eAsfivirus, Asfarviridae\u003c/em\u003e family[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Based on the 3' terminal sequence of the \u003cem\u003eB646L\u003c/em\u003e gene encoding the major capsid protein p72, ASFV was classified into 24 different genotypes. The virion is about 260 nm in diameter and has an icosahedral structure, and the viral genome is 170\u0026ndash;193 kb and encodes more than 150 open reading frames[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although the gene functions of many viral major proteins have been extensively studied[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the same gene can still exhibit different virulence across different viral strains. For example, deletion of \u003cem\u003eI267L\u003c/em\u003e in the ASFV-GS isolate results in loss of virulence in pigs, while this effect is not observed in the ASFV SY18 isolate[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This indicates that the gene functions in ASFV are highly intricate. Investigating the functions of the same gene in different viral strains will contribute to the development of live attenuated vaccines against ASF.\u003c/p\u003e \u003cp\u003eSince the occurrence of ASF in China in 2018[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], it has been reported in several other Asian countries, such as South Korea[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and Mongolia[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Due to the lack of safe and effective vaccines, ASF has spread widely and continues to evolve. In 2023, highly lethal genotype I and II recombinant strains were discovered in China, and attenuated live vaccine derived from genotype II cannot protect against the recombinant strains infection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, it is necessary to develop vaccines targeting recombinant strains.\u003c/p\u003e \u003cp\u003eThe development of gene deleted live attenuated vaccines against ASF has made notable progress. Single gene-deletions, including \u003cem\u003eI73R\u003c/em\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], \u003cem\u003eI177L\u003c/em\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cem\u003eMGF505-7R\u003c/em\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], \u003cem\u003eA137R\u003c/em\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and \u003cem\u003eI226R\u003c/em\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], have been shown to provide complete protection against genotype II virulent challenge in experimental models. Nevertheless, the safety profiles of such monogenic mutants warrant careful evaluation. Similarly, multigene deletion strains face challenges such as reduced viral replication and suboptimal immunogenicity, which may limit their protective efficacy[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recent advances in gene-deleted live attenuated vaccines have been comprehensively reviewed by Niu et al. (2025), who summarized the key virulence-related genes and discussed the prospects and challenges for ASFV vaccine development[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It is reported that immunization with \u003cem\u003eA137R\u003c/em\u003e or \u003cem\u003eI226R\u003c/em\u003e deleted attenuated strains provides complete protection against parental genotype II ASFV challenge with a favorable safety profile. Beyond I226R and A137R, other viral proteins have also been identified as critical modulators of host innate immunity. For instance, the H240R protein suppresses IL-1β production by targeting NF-κB signaling [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and I267L inhibits RNA polymerase III-RIG-I-mediated innate immunity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Mechanistically, I226R protein is known to impair antiviral responses, likely through multiple mechanisms including the suppression of NF-κB and IRF3 activation, to counteract innate immune responses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As a virulence-associated gene, \u003cem\u003eA137R\u003c/em\u003e inhibits cGAS-STING-mediated IFN-β production by promoting the autophagy-dependent lysosomal degradation of TBK1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, studies indicate that \u003cem\u003eA137R\u003c/em\u003e may also be implicated in mediating antibody-dependent enhancement (ADE) of infection [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Structural analysis reveals that \u003cem\u003eA137R\u003c/em\u003e forms a dodecahedral cage, potentially contributing to ASFV icosahedral capsid assembly[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCollectively, these findings suggest that, apart from \u003cem\u003eI177L\u003c/em\u003e, \u003cem\u003eA137R\u003c/em\u003e and \u003cem\u003eI226R\u003c/em\u003e represent promising targets for the development of live-attenuated ASF vaccines. However, most functional studies on these gene deletions have been conducted in the context of genotype II ASFV, and comparative analyses of their roles in other epidemiologically significant genotypes particularly in emerging genotype I/II recombinant strains is lacking, leaving their genotype-dependent effects on safety and immunogenicity unclear. Moreover, the protective efficacy and safety profile of a recombinant \u003cem\u003eI226R\u003c/em\u003e or \u003cem\u003eA137R\u003c/em\u003e deletion in a non-genotype II background remain unexplored. The global spread of ASF continues to threaten the swine industry, with sustained transmission in endemic regions and sporadic incursions into previously disease-free areas [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The development of safe and effective vaccines is therefore a global priority. Therefore, to address these gaps, this study was designed to generate and compare isogenic mutants derived from the genotype I/II recombinant strain JX23-02, featuring single deletions of \u003cem\u003eI226R\u003c/em\u003e, as well as a double \u003cem\u003eA137R\u003c/em\u003e/\u003cem\u003eI226R\u003c/em\u003e deletion mutant. We aimed to systematically evaluate the impact of these deletions on viral replication, attenuation, safety, and protective efficacy within this novel recombinant background. This work not only provides critical insights for vaccine design against emerging recombinant strains but also enriches our understanding of the genotype-specific functions of \u003cem\u003eA137R\u003c/em\u003e and \u003cem\u003eI226R\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cells and viruses\u003c/h2\u003e \u003cp\u003ePorcine alveolar macrophages (PAMs) and bone marrow-derived macrophages (BMDMs) were isolated from the bronchoalveolar lavage fluid and bone marrow aspirates of approximately 2-month-old pigs. The lavage fluids were supplemented with RPMI 1640 medium containing ethylenediaminetetraacetic acid (EDTA) and levofloxacin. After centrifugation, red blood cells were lysed using a red blood cell lysis buffer. The resultant cells were then washed three times with phosphate-buffered saline (PBS) containing levofloxacin and 2% fetal bovine serum (FBS), followed by resuspension in RPMI 1640 medium containing levofloxacin and 10% FBS. For the BMDMs, 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) was added to the resuspension. The cells were cultured for 7 days at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in the presence of GM-CSF before use.\u003c/p\u003e \u003cp\u003eASFV strain JX23-02 (GenBank accession: PP712069), isolated from pig spleen in 2023, is a genotype I/II recombinant variant and is preserved in our laboratory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Plasmid design for traditional recombination\u003c/h2\u003e \u003cp\u003eThe pMD18-T vector was used as the backbone. An EGFP expression cassette was inserted into the cloning site of pMD18-T. This cassette consisted of: a 1,258 bp left homology arm located upstream of the \u003cem\u003eI226R\u003c/em\u003e open reading frame (ORF), the p72 promoter, the enhanced green fluorescent protein (EGFP) gene, an SV40 poly(A) termination signal, and a 1,114 bp right homology arm located downstream of the \u003cem\u003eI226R\u003c/em\u003e ORF. To preserve the integrity of the \u003cem\u003eI243L\u003c/em\u003e gene during deletion of I226R, a 22-bp sequence from the 3' end of the \u003cem\u003eI226R\u003c/em\u003e gene was retained.\u003c/p\u003e \u003cp\u003eSeparately, an mCherry expression cassette was inserted into the cloning site of pMD18-T. This cassette comprised: a 1,061-bp left homology arm located upstream of the \u003cem\u003eA137R\u003c/em\u003e ORF, the p72 promoter, the mCherry fluorescent protein gene, an SV40 poly(A) signal, and a 1,238 bp right homology arm located downstream of the \u003cem\u003eA137R\u003c/em\u003e ORF.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Construction of gene-deletion JX23-02ΔI226R and JX23-02ΔI226RΔA137R\u003c/h2\u003e \u003cp\u003eUsing the homologous recombination method, the \u003cem\u003eI226R\u003c/em\u003e gene was replaced with EGFP gene to construct the \u003cem\u003eI226R\u003c/em\u003e gene-deletion recombinant in JX23-02. Two micrograms of the recombinant plasmid, which contains the left and right homologous arms and the p72 promoter-operated EGFP gene, were transfected into BMDMs in a 12-well plate. After 6 hours, the parental strain was inoculated at a multiplicity of infection (MOI) of 1.0. Upon the emergence of fluorescent cells, a single one was picked under a microscope for isolation, followed by separating the gene-deletion strain from the parental strain using the method of limited dilution. PCR was performed to confirm the absence of the parental virus in the purified JX23-02ΔI226R strain. If the parental strain JX23-02 was present, an 819 bp band would be amplified using the following primers: forward: 5\u0026rsquo;-CCAATAGGCAACTTTCTTTTG\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026rsquo;; reverse: 5\u0026rsquo;-ACAGGATAACGATGCCCTTA-3\u0026rsquo;.\u003c/p\u003e \u003cp\u003eUsing the same method, a dual-gene deletion virus JX23-02ΔI226RΔA137R was constructed by transfecting a recombinant expression plasmid containing about the \u003cem\u003eA137R\u003c/em\u003e gene left and right homologous arms and the p72 promoter-operated \u003cem\u003emCherry\u003c/em\u003e gene. After transfection, the purified JX23-02ΔI226R was inoculated at an MOI of 1.0. The recombinant virus was purified by picking individual fluorescent cells and employing limited dilution methods. Identification of the recombinant was performed by PCR, which amplified a 389 bp band using the following primers: forward: 5\u0026rsquo;-CAGTTCTTACCAAACTCGACC-3\u0026rsquo;; reverse: 5\u0026rsquo;-CATCTTGCCGATGAGATTTC-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Viral growth curve assay\u003c/h2\u003e \u003cp\u003eTo compare the growth curve of JX23-02, JX23-02ΔI226R, JX23-02ΔI226RΔA137R in Porcine alveolar macrophages (PAMs), each virus was inoculated at an MOI of 0.1 in triplicate of PAMs. After incubation for 1.5h, cells were washed with phosphate-buffered saline (PBS) and samples were collected at 24, 48, 72, 96, 120 h after infection, respectively. Subsequently, PAMs were used to detect the tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e) of virus solution at different time points.\u003c/p\u003e \u003cp\u003eThe viral titer was detected by the tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e) method and the direct immunofluorescence method. PAMs were resuscitated and then seeded into 96-well plates. Twelve hours later, the virus stocks at passages P2-P5 were repeatedly frozen and thawed, followed by 10-fold serial dilutions (from 10\u003csup\u003e1.0\u003c/sup\u003e to 10\u003csup\u003e8.0\u003c/sup\u003e). Subsequently, 100 \u0026micro;l of the diluted virus solution per well was inoculated into the 96-well plates. The cells were continuously cultured for 96 hours under the conditions of 37\u0026deg;C and 5% CO₂. The cells were then fixed with 4% paraformaldehyde and placed at 4\u0026deg;C for 20 minutes. After washing the cells with phosphate-buffered saline (PBS), 0.1% Triton X-100 was added and the cells were treated for 10 minutes. The cells were washed again with PBS, and then the diluted FITC-conjugated p30 monoclonal antibody (at a dilution ratio of 1:500) was added. The cells were incubated at 37\u0026deg;C in the dark for 60 minutes. After that, the cells were washed three times with PBST (0.05% Tween 20), with each washing lasting for 3 minutes. The washing solution was discarded, and the number of fluorescent wells was observed and counted under a fluorescence microscope. Finally, the viral titer was calculated according to the Reed-Muench method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Animal experiment\u003c/h2\u003e \u003cp\u003eTo explore the pathogenicity of genotype I/II recombinant strains in pigs, 18 Landrace pigs were randomly divided into four groups. Group A (061, 023, 042, 001, 091), Group B (100, 004, 007, 006, 066) and Group C (275, 276, 277, 278, 279) were the infection groups, with 5 pigs in each group. Group D (901, 902, 903) was the control group, consisting of 3 pigs. Pigs in the first three groups were inoculated intramuscularly with 1 mL of ASFV SY18, 1 mL of HuB20 and 1 mL of JX23-02 respectively in the neck, all at a titer of 100 TCID\u003csub\u003e50\u003c/sub\u003e. Pigs in Group D were injected with 1 mL of PBS as a blank control group. The rectal temperatures were measured daily, and their feeding and mental status were observed. If the infected pigs showed severe clinical signs, they were humanely euthanized. The observation lasted for 28 days.\u003c/p\u003e \u003cp\u003eTo assess the effects of deleting the \u003cem\u003eI226R\u003c/em\u003e gene and both the \u003cem\u003eI226R\u003c/em\u003e and \u003cem\u003eA137R\u003c/em\u003e genes on the virulence of the parental JX23-02 strain, 15 Landrace pigs were acquired from local farms for the experiment. The first group of 5 pigs (810,812,813,817,818) was intramuscularly inoculated with 10\u003csup\u003e5.0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of JX23-02ΔI226R, while another group of 5 pigs (081,082,083,084,085) was immunized with 10\u003csup\u003e5.0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of JX23-02ΔI226RΔA137R. The third group of 5 pigs (292,293,294,261,262) served as the challenge control. After inoculation, the clinical signs of the animals were monitored on a daily basis. For the surviving pigs during the immunity observation period, a challenge infection was performed via intramuscular injection of the parental strain ASFV JX23-02 at a dose of 100 TCID₅₀. Rectal temperature was taken daily, and oral and rectal swabs as well as blood samples were collected for the detection of viral nucleic acid and antibody levels in 0,3,7,10,14,17,21 days after inoculation and in 0,3,7,10,14,17,21,24,28 days after challenge. A part of the blood samples was preserved in EDTA blood collection tubes for viral load detection, and the other part was used for serum separation to detect antibodies. During the observation period, animals with severe clinical signs were humanely euthanized and collected the tissue samples including the heart, liver, spleen, lung, kidney, stomach, lymph nodes (submandibular and inguinal), thymus, tonsils, jejunum, ileum, colon, muscle, bone marrow, and quantitative polymerase chain reaction (qPCR) was carried out on tissue samples. The animal experiments were conducted in biosafety level 3 (BSL-3) laboratories.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Quantification of the ASFV load\u003c/h2\u003e \u003cp\u003eThe quantification of p72 gene copies in blood, oral swabs, anal swabs, and tissues was conducted using qPCR protocols recommended by the World Organization for Animal Health (WOAH) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.woah.org/en/home/\u003c/span\u003e\u003cspan address=\"https://www.woah.org/en/home/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Viral copy numbers were determined by qPCR using a standard curve based on the p72 gene, as previously described[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Tissue samples were cut into approximately 1 cm\u0026sup3; pieces and placed into sterile tubes containing steel beads. Subsequently, 1 mL of PBS was added, and the tissues were homogenized at 60 oscillations/s for 30 s using a tissue homogenizer; this cycle was repeated three times with 10-s intervals between cycles. The homogenates were then centrifuged at 12,000\u0026times;g for 5 min at 4\u0026deg;C. The supernatants were collected, transferred to new sterile microcentrifuge tubes, and labeled for subsequent use.\u003c/p\u003e \u003cp\u003eBriefly, DNA was extracted from each sample using an animal virus DNA extraction kit. Quantitative PCR (qPCR) was performed using the Premix Ex Taq (Probe qPCR) kit. The primers and probes used are listed in the additional table 1. The copy numbers of ASFV genomic DNA in samples were calculated based on a standard curve established using a standard plasmid containing the ASFV \u003cem\u003eB646L\u003c/em\u003e gene. The standard curve for the ASFV p72 gene in the quantitative PCR was fitted to the equation Y= \u0026minus;\u0026thinsp;3.245X\u0026thinsp;+\u0026thinsp;38.439, where Y represents the Ct value and X represents the copy number of the p72 gene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7\u003cb\u003eAntibody detection\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe indirect ELISA method, developed in our laboratory, was used to detect the level of ASFV p54 specific antibodies in serum. The ELISA plate was coated with p54 antibody and incubated overnight at 4\u0026deg;C. After blocking, a 50-fold diluted sample serum was added and incubated at 37\u0026deg;C for 30 minutes. The plate was washed five times with PBS, then incubated with Horseradish peroxidase (HRP)-labeled sheep anti-pig IgG at 37\u0026deg;C for 30 minutes. Following the wash, 80 \u0026micro;l of chromogenic solution 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to each well, incubated at room temperature in the dark for 10 minutes, then the reaction was stopped with 2 M sulfuric acid, and read within 3 minutes. Each serum sample was tested three times, and their absorbance values were measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 9.5 software was used for graphing and statistical analysis. The t-test was employed for significance analysis. \"ns\" indicates no significant difference, \"*\" represents a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), \"**\" indicates an extremely significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and \"***\" represents an extremely significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The pathogenicity of JX23-02.\u003c/h2\u003e \u003cp\u003eGeorgia07-like genotype II strain SY18 is highly lethal strain and HuB20 is naturally attenuated genotype II strain.To determine genotype I/ II recombinant strain virulence with genotype II, pigs were infected with the strains of JX23-02, HuB20 and SY18 at a dose of 100 TCID\u003csub\u003e50\u003c/sub\u003e respectively and measured the rectal temperature daily. Pigs in the control group remained clinically normal throughout the experiment. These three strains demonstrated a 100% mortality rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Among them, the pigs infected with the JX23-02 strain developed fever as early as 2 days post-infection (dpi), and the fever lasted for 1 to 3 days, with the earliest acute death occurring at 5 dpi. The pigs infected with the SY18 strain developed fever at 3 dpi, the fever persisted until around 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and showed signs of being on the verge of death as early as 7 dpi. The pigs infected with the HuB20 strain developed fever at 11 dpi, which lasted for about 10 days, and started to die as early as 22 dpi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Construction and growth characteristics of JX23-02ΔI226R and JX23-02ΔI226RΔA137R deletion strains.\u003c/h2\u003e \u003cp\u003eThe construction strategy of the ASFV gene-deleted strain is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, where the \u003cem\u003eI226R\u003c/em\u003e gene was replaced with the p72-EGFP cassette via homologous recombination. Since the terminal ends of the \u003cem\u003eI226R\u003c/em\u003e and \u003cem\u003eI243L\u003c/em\u003e open reading frames (ORFs) share 4 base pairs, when deleting the \u003cem\u003eI226R\u003c/em\u003e ORF, it is necessary to avoid disrupting the \u003cem\u003eI243L\u003c/em\u003e ORF. Therefore, 673 base pairs of the \u003cem\u003eI226R\u003c/em\u003e were deleted to protect the integrity of the \u003cem\u003eI243L\u003c/em\u003e ORF. After transfection and infection of BMDMs, the fluorescent cell lesions were collected. After multiple rounds of limiting dilution, virus expressing green fluorescence was purified and named JX23-02ΔI226R(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Routine polymerase chain reaction (PCR) was performed on the purified virus using differential primers, and no band of 819 base pairs in size was generated, indicating that the purification was successful (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The \u003cem\u003eA137R\u003c/em\u003e gene was completely deleted based on the JX23-02ΔI226R strain, and the \u003cem\u003eA137R\u003c/em\u003e gene locus was replaced with the p72-mCherry cassette. After purification, a recombinant virus emitting both red and green fluorescence was obtained and designated as JX23-02ΔI226RΔA137R (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Through PCR identification, the purified JX23-02ΔI226RΔA137R strain was obtained(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the effects of I226R and A137R deletion on ASFV JX23-02 replication, we evaluated the replication kinetics of JX23-02ΔI226R and ASFV JX23-02ΔI226RΔA137R in BMDMs. JX23-02ΔI226R or ASFV JX23-02ΔI226RΔA137R was infected at an MOI of 0.01, and the virus yields were detected at 0, 24, 48, 72, 96, and 120 hours post-infection (hpi), respectively. The results showed that compared with the parental strain, the viral titers of the recombinant strains JX23-02ΔI226R and JX23-02ΔI226RΔA137R were significantly reduced at 24 hpi, 48 hpi, and 72 hpi (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). At 96 hpi and 120 hpi, the viral titers of the parental strain were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those of the double gene-deleted strain JX23-02ΔI226RΔA137R, while there was no significant difference in the titers between the single gene-deleted strain JX23-02ΔI226R and the parental strain at 120 hpi (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In addition, the titers of the double gene-deleted strain JX23-02ΔI226RΔA137R were slightly lower than those of the single gene-deleted strain JX23-02ΔI226R at 72 hpi and 96 hpi, but there was no statistically significant difference between them (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Overall, the deletion of the \u003cem\u003eI226R\u003c/em\u003e and \u003cem\u003eA137R\u003c/em\u003e genes had a stage-specific effect on the in vitro replication ability of the ASFV JX23-02 strain, especially during the middle stage of infection (24\u0026ndash;72 hpi). The specific results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 JX23-02ΔI226R exhibits residual virulence but provides protection in surviving pigs.\u003c/h2\u003e \u003cp\u003eJX23-02ΔI226R attenuated part of the virulence of the parental virus strain and caused the death of some pigs. However, the deletion strain also provided complete protection to the surviving pigs against the challenge of the parental virus strain.\u003c/p\u003e \u003cp\u003eFive pigs were immunized intramuscularly with a dose of 10\u003csup\u003e5.0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of JX23-02ΔI226R. Three pigs (810, 817, 818) died on days 11, 12, and 16 post-immunization, respectively. The deceased pigs exhibited typical clinical signs of ASF, including fever, depression, lethargy, and anorexia, with peak body temperatures reaching 41.5\u0026deg;C [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The remaining two pigs showed normal performance within the 21-day observation period, resulting in a survival rate of 40% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The pigs started to develop fever on the third day after immunization, with the fever reaching its peak from the 7\u0026ndash;10 dpi and lasting for 5\u0026ndash;13 days. Two pigs exhibited pyrexia, with individual peak temperatures reaching 40.9\u0026deg;C and 40.8\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The febrile response was accompanied by anorexia and reduced mobility, and both animals recovered fully within 3\u0026ndash;4 days after symptom onset.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e21 days after immunization, the pigs were challenged intramuscularly with a dose of 100 TCID\u003csub\u003e50\u003c/sub\u003e. The body temperatures of the two surviving pigs immunized with JX23-02ΔI226R increased transiently on 7 to 9 post-challenge (dpc) and then returned to normal(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). They survived with normal physiological performance within the 28-day observation period. In the control group, the body temperatures of the pigs increased sharply on the 3rd day after challenge, and died at 5 dpc. All pigs in the control group died within 8 dpc (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 JX23-02ΔI226RΔA137R can be completely attenuated and provides complete protective efficacy against the parental virus strain\u003c/h2\u003e \u003cp\u003eJX23-02ΔI226RΔA137R is completely attenuated relative to the parental virus strain and can provide full protection to immunized pigs. Five pigs were immunized intramuscularly with a dose of 10\u003csup\u003e5.0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of JX23-02ΔI226RΔA137R. All pigs survived during the immunization observation period and also after the challenge with the parental virus strain. The pigs started to have a slight fever on the 5th day after immunization, which lasted for 1\u0026ndash;3 days. There was some fluctuation in body temperature during the observation period, but the maximum body temperature did not exceed 40.1\u0026deg;C. 21 days after immunization, the five pigs were challenged with 100 TCID\u003csub\u003e50\u003c/sub\u003e of the parental virus strain JX23-02. All pigs survived, with their body temperatures remaining basically normal and no obvious fever symptoms shown.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 Occasional shedding occurs through the oral or anal route after JX23-02ΔI226R and JX23-02ΔI226RΔA137R inoculation and ASFV JX23-02 challenge.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDuring the observation period after immunization and challenge, oral and anal swabs were collected from pigs, DNA was extracted, and viral load was detected by qPCR. The detection results of oral and anal swabs after immunization are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In the pigs immunized with JX23-02ΔI226R, the oral and anal swabs showed positive results in 4 pigs during the early stage (within 7 to 14 days) after vaccination, and viremia was detected in all these pigs. In contrast, in the pigs immunized with JX23-02ΔI226RΔA137R, all swab samples tested negative.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe amplification results of the p72 gene on the oral and anal swabs after challenge infection showed that in the control group pigs, viral shedding was detected starting from 3 days after challenge, and the copy number of viral DNA was relatively high. In the oral and anal swabs of the JX23-02ΔI226R immunized group, viral shedding was detected in 1 to 2 pigs. In the JX23-02ΔI226RΔA137R immunized group, viral DNA was only detected in the anal swab of one pig, showing a weakly positive result. This suggests that JX23-02ΔI226RΔA137R may have an improved safety profile compared to JX23-02ΔI226R.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 JX23-02ΔI226RΔA137R induces lower viremia and tissue viral load compared to JX23-02ΔI226R\u003c/h2\u003e \u003cp\u003eWe performed qPCR on the whole blood of pigs collected after immunization and challenge to detect the viral genome content. Autopsies were carried out on each dead pig. All organs of the dead pigs in the control group and the JX23-02ΔI226R immunization group showed typical gross pathological changes of African swine fever, such as splenomegaly and visceral hemorrhage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA.5C). Aside from a few petechial hemorrhages observed in the lungs, no obvious pathological changes were observed in the pigs of the JX23-02ΔI226RΔA137R immunization group. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Organ tissue samples were collected for quantitative detection of the viral genome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the blood samples after immunization, viral DNA was detected at high levels in the blood of control group pigs after challenge, and the viral DNA copy number in various tissues was also high. The viral content in the blood of pigs immunized with JX23-02ΔI226R was significantly higher than that in the JX23-02ΔI226RΔA137R group. The viral genome in the blood was undetectable (negative) by qPCR 28 days after challenge. In the JX23-02ΔI226R group, the viral load in the tissues of the dead pigs after immunization was high, and qPCR was positive in all types of tissue samples. In the surviving pigs, the virus was detected in the colon, jejunum, and bone marrow, indicating that the virus in the tissues and organs could not be completely cleared.\u003c/p\u003e \u003cp\u003eIn the JX23-02ΔI226RΔA137R group, viremia disappeared 21 days after challenge, and the blood qPCR test was negative. In the detection of the viral genome in tissue samples, the kidney, inguinal lymph node, thymus, and stomach tissues tested negative, while the viral genome was detected in other tissues, but only a limited number of samples tested positive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 JX23-02ΔI226R and JX23-02ΔI226RΔA137R induced strong immune response\u003c/h2\u003e \u003cp\u003eThe kinetics of p54-specific antibody production were consistent with those typically observed for attenuated ASFV vaccines. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, antibody levels were detectable in all experimental groups on day 7 post-vaccination, peaking approximately between days 17 and 21 post-vaccination. Following challenge, the p54 antibody levels in the experimental groups slightly increased and then declined moderately over time, but remained at a high positive level throughout the observation period. In contrast, p54 antibodies were only detected at a low level in the control group after challenge.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn the present study, we successfully constructed a single gene-deleted strain (JX23-02ΔI226R) and a double gene-deleted strain (JX23-02ΔI226RΔA137R) derived from the genotype I/II recombinant ASFV strain JX23-02. The emergence of genotype I/II recombinant ASFV strains poses new challenges for vaccine development, as existing genotype II-based vaccines have shown limited protection against these variants [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Our successful attenuation of the recombinant strain JX23-02 through gene deletion demonstrates that targeted modification of circulating field strains is a feasible strategy to address this challenge. Subsequently, we systematically evaluated their immunogenicity, protective efficacy, and safety as potential vaccine candidates. A key finding of this study is that the deletion of \u003cem\u003eI226R\u003c/em\u003e and \u003cem\u003eA137R\u003c/em\u003e genes significantly impaired the in vitro replication capacity of ASFV, particularly during the middle stage of infection (24\u0026ndash;72 h), as evidenced by a marked reduction in viral titer. This observation provides critical theoretical insights into the development of attenuated ASFV vaccines, highlighting the potential of \u003cem\u003eI226R\u003c/em\u003e and \u003cem\u003eA137R\u003c/em\u003e as key virulence-related targets for viral attenuation.\u003c/p\u003e \u003cp\u003eAnimal experiments revealed distinct levels of immune protection among the constructed strains. Specifically, JX23-02ΔI226R retained residual virulence, which led to the death of some immunized pigs; however, it still induced effective protective immune responses against the parental strain, indicating its potential as a vaccine candidate with room for further optimization. In contrast, JX23-02ΔI226RΔA137R exhibited superior safety profiles: all immunized pigs survived the challenge without manifesting obvious clinical signs. This result demonstrates that the additional deletion of the \u003cem\u003eA137R\u003c/em\u003e gene further attenuated viral virulence without compromising immunogenicity, confirming the synergistic effect of dual gene deletion on ASFV attenuation. Furthermore, qPCR analysis showed that pigs immunized with JX23-02ΔI226RΔA137R had significantly shorter duration of viremia and lower viral load compared to those in the JX23-02ΔI226R group. These data further validate the advantages of JX23-02ΔI226RΔA137R in suppressing viral replication and reducing viremia persistence, thereby providing robust support for its potential as a promising vaccine candidate. The enhanced safety profile of JX23-02ΔI226RΔA137R may be attributed to the cumulative effect of deleting two immunomodulatory genes. \u003cem\u003eI226R\u003c/em\u003e suppresses NF-κB and IRF3 activation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], while A137R inhibits cGAS-STING-mediated IFN-β production by promoting autophagy-dependent degradation of TBK1 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Simultaneous deletion of both genes likely leads to more robust innate immune responses against the virus, thereby limiting its replication and dissemination in vivo, which explains the reduced viremia and lower tissue viral load observed in this group. Collectively, based on reduced viremia, lower tissue viral load, and absence of mortality, JX23-02ΔI226RΔA137R outperformed JX23-02ΔI226R in terms of safety, immunogenicity, and viral replication control, offering valuable experimental basis and theoretical support for the development of ASFV vaccines.\u003c/p\u003e \u003cp\u003eOur findings exhibit both similarities and discrepancies with previous reports on ASFV gene-deleted vaccines. Zhang et al.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] demonstrated that the SY18ΔI226R strain did not induce obvious clinical signs after vaccination and conferred complete protection against lethal challenge with a virulent strain. While JX23-02ΔI226R in the present study also provided partial protective efficacy, its higher residual virulence resulted in partial mortality among immunized animals. This discrepancy is likely attributed to the inherent genetic background differences and distinct pathogenic mechanisms between the parental strains (SY18 and JX23-02), emphasizing the importance of tailoring attenuation strategies to specific ASFV genotypes or recombinant strains. On the other hand, Zhao et al. reported that the genotype II attenuated live vaccine HLJ/18-7GD failed to protect against genotype I/II recombinant ASFV strains, suggesting that genomic recombination of ASFV may lead to the emergence of novel epitopes or immune escape mechanisms, thereby compromising the protective efficacy of existing vaccines [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The successful development of JX23-02ΔI226RΔA137R in this study addresses this challenge by proposing a multi-gene deletion strategy, which achieves enhanced attenuation while preserving critical immunogenic determinants. Recently, Peng et al. demonstrated that deletion of CD2v and A137R from another genotype II strain also resulted in complete attenuation and protection [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. While their double-deletion strategy targeted different genes (CD2v and A137R), our results with JX23-02ΔI226RΔA137R similarly confirm that A137R is a key target for attenuation across different genetic backgrounds, and suggest that multiple gene combinations can achieve optimal safety and efficacy.\u003c/p\u003e \u003cp\u003eIn conclusion, we successfully constructed two gene-deleted strains (JX23-02ΔI226R and JX23-02ΔI226RΔA137R) based on the genotype I/II recombinant ASFV strain JX23-02. Deletion of \u003cem\u003eI226R\u003c/em\u003e and \u003cem\u003eA137R\u003c/em\u003e significantly attenuated viral replication capacity. Among the candidates, JX23-02ΔI226RΔA137R exhibited excellent performance in safety, immunogenicity, and protective efficacy, whereas JX23-02ΔI226R induced partial protective immunity but retained undesirable residual virulence. These results highlight the feasibility of multi-gene deletion as a promising approach for the development of safe and effective ASFV vaccines, especially against emerging recombinant strains.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences (protocol number: IACUC of AMMS-11-2024-67) and were conducted in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe ASFV strain JX23-02 genome sequence is available in GenBank under accession number PP712069.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work is financially supported by the Foundation of State Key Laboratory of Pathogen and Biosecurity (grant No. SKLPBS2421) and the National Key Research and Development Program of China (Grant No. 2021YFD1800100). The funding body had no role in the design of the study, data collection, analysis, interpretation, or writing of the manuscript.\u003c/p\u003e\n\u003cp\u003eAuthors' contributions\u003c/p\u003e\n\u003cp\u003eConceptualization: HZ, RH, SZ, FM; methodology: HZ, QL, NL, TH; validation: RH, FM; data collection: HZ, QL; writing—original draft preparation: HZ; writing—review and editing: QL, NL, TH, HW, RH, SZ, FM; supervision: RH, SZ, FM. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003evan den Born E., Olasz F., M\u0026eacute;sz\u0026aacute;ros I., G\u0026ouml;ltl E., Ol\u0026aacute;h B., Joshi J., van Kilsdonk E., Segers R., Z\u0026aacute;dori Z., African swine fever virus vaccine strain Asfv-G-∆I177l reverts to virulence and negatively affects reproductive performance, NPJ Vaccines. 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(2020) 8:221-246.\u003c/li\u003e\n \u003cli\u003eZuo X., Peng G., Xia Y., Xu L., Zhao Q., Zhu Y., Wang C., Liu Y., Zhao J., Wang H., Zou X., A quadruple fluorescence quantitative PCR method for the identification of wild strains of african swine fever and gene-deficient strains, Virol J. (2023) 20:150.\u003c/li\u003e\n \u003cli\u003eZuo X., Peng G., Zhao J., Zhao Q., Zhu Y., Xu Y., Xu L., Li F., Xia Y., Liu Y., Wang C., Wang Z., Wang H., Zou X., Infection of domestic pigs with a genotype II potent strain of ASFV causes cytokine storm and lymphocyte mass reduction, Front Immunol. (2024) 15:1361531.\u003c/li\u003e\n \u003cli\u003eZhang Y., Ke J., Zhang J., Yang J., Yue H., Zhou X., Qi Y., Zhu R., Miao F., Li Q., Zhang F., Wang Y., Han X., Mi L., Yang J., Zhang S., Chen T., Hu R., African Swine Fever Virus Bearing an I226R Gene Deletion Elicits Robust Immunity in Pigs to African Swine Fever, J. Virol. (2021) 95:e0119921.\u003c/li\u003e\n \u003cli\u003eZhao D., Sun E., Huang L., Ding L., Zhu Y., Zhang J., Shen D., Zhang X., Zhang Z., Ren T., Wang W., Li F., He X., Bu Z., Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs, Nat Commun. (2023) 14:3096.\u003c/li\u003e\n \u003cli\u003ePeng G., Zhao X., Zou X., Zhang H., Zhao J., Zuo X., Tan S., Wu R., Guan X., Li S., Xu Y., Xia Y., Xu X., Xu L., Zhu Y., Liu J., Liu Y., Gao G.F., An attenuated African swine fever virus with deletions of the CD2v and A137R genes offers complete protection against homologous challenge in pigs, J Virol. (2025) 99:e0026225.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"African swine fever virus, Gene deletion, I226R, A137R, Recombinant strain","lastPublishedDoi":"10.21203/rs.3.rs-9205759/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9205759/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: The emergence of highly lethal genotype I/II recombinant African swine fever virus (ASFV) strains in China has rendered existing genotype II-based live attenuated vaccines ineffective, underscoring the urgent need for novel vaccine candidates. Although single-gene deletions of \u003cem\u003eA137R\u003c/em\u003e or \u003cem\u003eI226R\u003c/em\u003e have shown promise against genotype II strains, their efficacy and safety in the context of recombinant strains remain unexplored.\u003c/p\u003e\n\u003cp\u003eMethods: Using homologous recombination, we constructed a single gene-deleted mutant (JX23-02ΔI226R) and a double gene-deleted mutant (JX23-02ΔI226RΔA137R) from the genotype I/II recombinant ASFV strain JX23-02. The replication kinetics, pathogenicity, immunogenicity, and protective efficacy of these mutants were evaluated in vitro and in pigs.\u003c/p\u003e\n\u003cp\u003eResults: Both deletion mutants exhibited significantly reduced replication in porcine alveolar macrophages. Immunization with JX23-02ΔI226R resulted in 40% survival, but surviving pigs were fully protected against subsequent lethal challenge. In contrast, JX23-02ΔI226RΔA137R was completely attenuated: all immunized pigs survived challenge without any clinical signs and developed robust p54-specific antibody responses. Moreover, viral shedding and tissue viral loads were markedly lower in the double-deletion group than in the single-deletion group.\u003c/p\u003e\n\u003cp\u003eConclusions: The double gene-deleted mutant JX23-02ΔI226RΔA137R represents a safe and efficacious live attenuated vaccine candidate against genotype I/II recombinant ASFV strains, highlighting the superiority of multi-gene deletion strategies for ASF vaccine development.\u003c/p\u003e","manuscriptTitle":"Evaluation of An I226R and A137R deletion mutant from a genotype I/II recombinant African swine fever virus strain as a live attenuated vaccine in pigs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-11 06:05:53","doi":"10.21203/rs.3.rs-9205759/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4db09299-f09c-4b04-8db7-8da8aef027af","owner":[],"postedDate":"April 11th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-07T05:50:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T18:01:24+00:00","index":16,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T05:55:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-11 06:05:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9205759","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9205759","identity":"rs-9205759","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}