MGF100 but not MGF300 family is a potential multigene-deleted target for ASFV attenuation and live attenuated vaccine development

MGF100 but not MGF300 family is a potential multigene-deleted target for ASFV attenuation and live attenuated vaccine development | 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 Article MGF100 but not MGF300 family is a potential multigene-deleted target for ASFV attenuation and live attenuated vaccine development Wen Dang, Fan Xu, Mingyang Ding, Tao Li, Yu Du, Huanan Liu, Zhengwang Shi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6859140/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 African swine fever is a highly contagious and lethal disease of swine, but there are few therapeutic options available for treatment. Therefore, it is urgent to develop safe and effective vaccines. In this regard, we described the differential effect of deletion of whole MGF100 and MGF300 families from Genotype Ⅱ highly virulent strain on virus replication, virulence and induction of protection. The resulting ASFV-Δ100 and ASFV-Δ300 mutants demonstrated reduced growth kinetics in vitro, with the former displaying aberrant virus morphogenesis. The ASFV-Δ100 was efficiently attenuated, whereas the ASFV-Δ300 still retained virulence. In homologous lethal challenge, two mutants achieved the same protection rate, with the former providing more pathological protection on organs. Mechanically, we found that ASFV-Δ100 was capable of inducing more robust innate immune in vitro and more consistent P30 antibody response in vivo. To conclude, the MGF100 family is a potential multigene-deleted target for ASFV live attenuated vaccine development. Biological sciences/Microbiology/Vaccines/Live attenuated vaccines Biological sciences/Immunology/Infectious diseases/Viral infection African swine fever virus Multigene family 100 Multigene family 300 Morphogenesis Innate immune Protective efficacy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction African swine fever (ASF) is a highly infectious and severe haemorrhagic viral disease of swine with high mortality. The disease is circulating on the African, European and Asian continents, as well as recently in Dominican Republic and Haiti 1 . As the only member of the Asfarviridae family, genus Asfivirus , the etiological agent ASF virus (ASFV) is a double stranded DNA virus wrapped in a multi-layer icosahedral structure with a diameter of about 200 nm and notably the only DNA virus (arbovirus) that can be transmitted through arthropods 2 . The spread of the virus continues, exacerbated by the fact that nowadays there are no effective therapeutic options against ASFV infection 3 . Vaccination is seemingly a promising tool, of which the live attenuated vaccines so far make the most progress in term of efficacy and protection, although some concerns are raised about the safety issues. The multigene families (MGFs) are mainly distributed at both ends of the viral genome and play important roles in affecting viral virulence, regulating interferon response and inflammatory response. MGFs are divided into five major categories, namely MGF100, MGF110, MGF300, MGF360 and MGF505 4 . MGFs have always been good options for deletion as a way of virus attenuation, in which combinational deletions of MGF360 and MGF505 genes are well characterized. The virulent Genotype Ⅱ Georgia 2007/1 isolate harboring combinational deletions of MGF360 and MGF505 genes is highly attenuated in swine and is able to provide protection against homologous challenge 5 . In parallel, the same deletions exerted same effects on Genotype Ⅰ virulent strain, as deletions of MGF360 genes (MGF360-10L, 11L, 12L, 13L and 14L) and MGF505 genes (MGF505-1R, 2R and 3R) and interrupting genes (MGF360-9L and MGF505-4R) in the genome of Benin 97/1 reduced its virulence in domestic pigs and induced a protective response 6 . Those data imply that multiple-gene deletions, especially deletions of MGFs are a promising way to produce an attenuation when there is no clear identification of the function and role of individual target genes (Table S12). Compared to the MGF110, MGF360 and MGF505 families, the MGF100 and MGF300 families are relatively less studied. So far, the role of the individual genes from MGF100 and MGF300 families is poorly understand. It is empirically suggested that compared to single-gene deletions, multiple-gene deletions are more capable of attenuating ASFV and developing live attenuated candidate. In this regard, two ASFV candidates were developed based on the highly virulent Genotype Ⅱ Eurasian strain ASFV CN/GS/2018 with one losing the MGF100 family and the other losing the MGF300 family, namely ASFV-Δ100 and ASFV-Δ300, respectively. Later the pathogenicity, immunogenicity and protective response of two candidates were compared in animal experiment. We find that ASFV-Δ100 is able to find a better balance in virulence, immunogenicity and protective response, as ASFV-Δ100 infection results in no or extremely slight ASFV-compatible clinical signs. On the contrary, ASFV-Δ300 is not completely attenuated, as evidenced by the death of 2 pigs following vaccination. Meanwhile, irrespective of same protection rates in the residual pigs following lethal challenge, the ASFV-Δ300 group demonstrated more severe post-mortem signs and immuno-histochemistry injury when compared to ASFV-ΔMGF100 group. At the same time, we also find that the virion morphogenesis changes significantly after deletion of MGF100-2R, showing a subset of forms, including curved, linear and semilunar membranes, which may partially explain the decline of virus virulence. RNA-sequencing data also display the fact that ASFV-Δ100 is capable of inducing more robust innate immune compared to ASFV-Δ300, which potentially uncovered its stronger protective capacity. In summary, MGF100 but not MGF300 family is a more promising multigene-deleted target for ASFV attenuation and live attenuated vaccine development. The ASFV-Δ100 is a promising live attenuated vaccine candidate, though further research on it is needed. Results Conservation and transcription kinetics of MGF100 and MGF300 families. The open reading frames (ORFs) of MGF100 family are located at positions 11094-11468 (MGF100-1R), 179519-179944 (MGF100-2L) and 180309-180617 (MGF100-3L) of the full-length genome sequence of ASFV CN/GS/2018. Likewise, four members of MGF300 family are located at positions 19732-20538 (MGF300-1L), 21365-21847 (MGF300-2R), 21937-22251 (MGF300-3L) and 21937-22929 (MGF300-4L). As MGF300-3L is a C-terminally truncated version of MGF300-4L, it is not further characterized in this study. A comparative study of conservation is performed to evaluate the conservation of MGF100 and MGF300 members across variants of ASFV Genotypes Ⅰ, Ⅱ, Ⅳ, Ⅴ (data resource: The African Swine Fever Virus Database). The amino acid identity of MGF100-1R is absolutely high across Genotype Ⅰ/Ⅱ strains, ranging from 94% to 100% (Fig. S1A, Table S1). Paralleling with the conservation results from MGF100-1R, MGF100-2L demonstrated the same trends. Of significant note, two Genotype Ⅱ variants with truncated version of MGF100-2L were observed, with the size of 48 amino acids in Ukraine_Kyiv_2014_QED21767.1 and 75 amino acids in Ukraine_Kyiv_2014_QED21766.1. Also, a relatively high conservation is observed, ranging from 95% to 97% in Genotype I strains and from 82% to 90% in other genotypes (Fig. S1B, Table S2). The situation in MGF100-3L is relatively different. Across most Genotype Ⅱ strains, a length size of 102 amino acids is dominantly observed with high identity. However, a longer length of 146 amino acids is observed in three Genotype Ⅱ strains, namely South_Africa_KNP_Pretorisuskop_1996, Malawi_Tengani_1962 and South_Africa_Warmbaths_1987. Surprisingly, Genotype I strains all carry a 146 amino-acid MGF100-3L protein with an identity of 84%. The amino acid identity in Genotype Ⅳ, Ⅶ, Ⅸ and Ⅺ strains is divergent, ranging from 75% to 97% (Fig. S1C, Table S3). In the context of MGF300-1L, the identity is high, ranging from 95% in Genotype I to 100% in Genotype Ⅱ (Fig. S2A, Table S4), which is the case for MGF300-2R (Fig. S2B, Table S5). Of note, there are two dominant versions of MGF300-4L, one is 254 amino acid length and the other is 330 amino acid length. Irrespective of the length, MGF300-4L is highly conserved in Genotype Ⅰ/Ⅱ strains but diverse in other genotype strains (Fig. S2C, Table S6). Previous study demonstrated the transcriptome profile of ASFV genes, highlighting distinctive patterns of individual gene expressions. Three members of MGF100 demonstrated the same transcriptional trends during ASFV infection. Like VP30, the expression level of MGF100 members peaked at the very early of 3 hours post infection (hpi), stayed at a plateau for the next 6 h and began to decline starting at 9 hpi (Fig. 1A). However, MGF300 members are divergent in transcriptional patterns. MGF300-2R and MGF300-4L demonstrated the same trends as MGF100. On the contrary, MGF300-1L is proved to be a late gene, as its expression continuously increased over time (Fig. 1C). The spatial localization of MGF100 and MGF300 in ASFV-infected cells is further characterized. By employing IFA in 293T cells, we found proteins encoded by MGF100 and MGF300 were dominantly localized in the cytoplasm, except for MGF300-1L, as it demonstrated a dominant distribution in the cytoplasm and a discrete distribution in the nuclear (Fig. 1B and D). Construction and characterization of ASFV-Δ100 and ASFV-Δ300 mutants. To understand how MGF100 family is involved in ASFV replication capacity in vitro and virulence in vivo , the three-gene-deleted ASFV-Δ100 mutant was constructed and further characterized (Fig. 2A). The precision of MGF100 family deletion was further validated by RT-PCR (Fig. 2A) and whole-genome sequencing (Fig. S1B). As anticipated, expected deletion was well achieved. Likewise, ASFV-Δ300 mutant was developed with the same strategy and demonstrated high efficiency of expected genomic modifications, as analyzed by RT-PCR (Fig. 2B) and whole-genome sequencing (Fig. S1C). However, apart from expected deletions, during the process of homologous recombination, some unexpected genomic modifications were observed, as summarized in Table S7 for ASFV-Δ100 and Table S8 for ASFV-Δ300. In depicting multi-step virus growth curves of two mutants, we found that ASFV-Δ100 and ASFV-Δ300 demonstrated the same trends in replication capacity during the first burst period, starting from 2 to 12 hpi. However, over time ASFV-Δ100 and ASFV-Δ300 viral loads were significantly reduced by approximately 0.5 log compared with ASFV-WT depending on the time points considered (Fig. 2C and E), indicating that MGF100 and MGF300 families are determinants for virus growth in vitro . The consistent results were also observed at the protein level (Fig. 2D and F). MGF100-2R is potentially involved in the assembly of ASFV particles. The MGF family is proved to be multifunctional in roles ranging from suppressing the type Ⅰ interferon response to virulence in pigs. However, the role of MGFs in involving in ASFV morphogenesis is poorly understood. Cells infected with ASFV-WT or MGF300-deleted ASFV clearly showed whole virions in either mature (Fig. 3A, black arrow) or immature (Fig. 3A, white arrow) stages surrounding membrane assembly intermediates. Particularly, an electron dense protein/DNA accumulation was observed in the center of the factory. Meanwhile, the partly formed icosahedral structures are commonly observed, with the capsid protein layer accumulating on the outer face and inner core shell proteins assembling on the inner face. However, in the cells infected with MGF100-deleted ASFV, strikingly, we found that the membrane assembly intermediates displayed a subset of forms, including curved, linear and semilunar membranes as well as membrane fragments devoid of the dense capsid or core shell protein layers (Fig. 3A, red arrow). To elucidate which member of MGF100 family is involved in ASFV morphogenesis, individual genes of the MGF100 family are deleted from the ASFV-WT. Our data suggests that deletion of MGF100-2L itself results in the changes of ASFV morphogenesis (Fig. 3B, red arrow), implying that MGF100-2L is involved in the assembly of ASFV particles, even though the exact mechanism is unclear. Evaluation of ASFV mutants virulence and protective efficacy in pigs. To evaluate the potential of MGF100 and MGF300 families as virulence factors whose mutants can be used as live attenuated vaccines, in vivo evaluation of safety, immunogenicity and protective efficacy was performed. In detail, domestic pigs weighing about 30 kg were intramuscularly (IM) vaccinated with ASFV-Δ100 (n = 7) or ASFV-Δ300 (n = 7)) at a dose of 10 6 .0 HAD 50 at which was lethal for pigs in the case of ASFV-WT. During a period of 21 days observation, a serial of clinical symptoms compatible with ASFV infection were monitored (Fig. 4A). All animals in the ASFV-Δ100 group survived and remained healthy, only two of which (No. 5400 and No. 5410) displayed recurrent transient fever at the very first beginning of vaccination (Fig. 4B and D). However, two animals (No. 5386 and No. 5433) in the ASFV-Δ300 group started to develop fever at 4 days post vaccination (dpv) and died on 13 and 16 dpv (Fig. 4B and F). The residual pigs are asymptomatic, however, remained a relatively worse health status, as evidenced by less food take and depression. Apparently, ASFV-Δ300 is partially attenuated and retained some level of virulence in pigs when compared to ASFV-Δ100. In the coming evaluation of immunogenicity and protective efficacy, vaccinated pigs were later challenged with a lethal dose (10 2.0 HAD 50 ) of ASFV-WT via IM route. To facilitate comparison between 2 mutants, five out of seven surviving pigs in the ASFV-Δ100 group were selected for the challenge experiment. Five naïve animals challenged with ASFV-WT virus started to develop fever at 4 days post challenge (dpc), peaked at 6 dpc and subsequently died within 9 dpc (Fig. 4C and E). On the contrary, 4 out of 5 pigs in ASFV-Δ100 and ASFV-Δ300 group survived the homologous lethal challenge, with one died on 9 dpc for the former group and one died on 13 dpc for the latter group (Fig. 4C, E and G; Table S9). To further observe the clinical presentations of animals during vaccination and challenge, virus shedding and viremia were further quantitatively assessed. Pigs in the ASFV-Δ100 group demonstrated fluctuating but relatively high level of viremia in the beginning of vaccination. The viremia tends to drop to low level at the end of the vaccination (18 - 20 dpv) (Fig. 5A). In the ASFV-Δ300 group, two dead pigs displayed high level of viremia, which was consistent with other clinical symptoms. For the surviving pigs, they tend to have comparable level of viremia as ASFV-Δ100 group (Fig. 5C). The situation of virus shedding in fecal and oral-nasal swabs is the same for both mutants (Fig. S5). During challenge period, surviving pigs in both groups developed the transient fever between 2 and 6 dpc but quickly regained the healthy status. Consistently, surviving pigs from both groups displayed a low or undetectable level of viremia and virus shedding, suggestive of good protection (Fig. 5B and D; Fig. S5), even the two dead pigs were immunogenic to high level of ASFV-WT replication. Those data primarily imply that ASFV-Δ300 is attenuated but more pathogenic when compared to ASFV-Δ100. ASFV-Δ100 demonstrated higher immunogenicity against homologous challenge. The underlying protective mechanism of live attenuated vaccine against ASFV infection is not fully unclear, however, it is strongly believed that antibody response is playing a key role. In the vaccination phase, ASFV-Δ100 group reach the peak of VP30 antibody at 12 dpv at a fast and uniform pace (Fig. 5E). In the ASFV-Δ300 group, the VP30 response is a little bit delayed and inconsistent among pigs, though all reached the plateau at the end of vaccination (Fig. 5G). At the period of challenge experiment, the VP30 response remained at the same plateau, except one dead animal from ASFV-Δ100 group demonstrated a slight decline of VP30 (Fig. 5F). On the contrary, all the naive pigs died before VP30 response was full activated. Those data imply that VP30 response is a potential marker for measuring the protective response exerted by live attenuated vaccine. ASFV-Δ100 is capable of providing more pathological protection on organs following lethal challenge. As the protection rate is the same for ASFV-Δ100 and ASFV-Δ300 in the animal challenge experiment, we were further prompted to compare the protection efficacy of both mutants at the pathological level. When detecting the viral loads in organ samples, it is well observed that viral loads in ASFV-Δ100 group is significantly lower when compared to the ASFV-Δ300 group and ASFV-WT group (Fig. 6A). Those findings primarily indicate that ASFV-Δ100 but not ASFV-Δ300 is providing more protection against homologous challenge. In search of more evidence to support our hypothesis, the postmortem data demonstrated pulmonary congestion, pneumonia with caseous necrosis (occasionally accompanied by local calcification), foaming of the trachea and bronchus, and severe alveolar and interstitial pulmonary oedema in dead pigs from control group (red arrow) 7 . Specifically, spleen is characterized with enlargement and friability accompanied by dark red or even black round edges (red arrow). Petechiation on the capsule of the kidney is obviously and frequently observed with severe ecchymoses (cortexes and pelves) (red arrow). Apart from that, hepatic congestion, gallbladder bleeding, lymph node enlargement and severe haemorrhage are also common in organs from dead pigs (Fig. 6B, red arrow). On the contrary, the presence of ASFV-compatible pathological lesions, was less observed in the ASFV-Δ100 and ASFV-Δ300 pigs. It seems that the severity of pathological lesions in both groups is greatly but equally relieved. To gain more evidence, histopathological observation of organ injury was further performed. Consistent with pathological findings, notably, the most obvious immunohistochemical changes associated with ASFV infection are multifocal interstitial hemorrhages, apoptosis/necrosis of cells and presence of moderate amounts of positively labeled cells consistent with histiocytes 8 . Particularly in pathology, organs infected with ASFV-WT virus showed large-scale interstitial neutrophilic infiltration (black arrow) and hemorrhage (green arrow), which in two immunized groups were no bleeding. The lymph nodes in the WT group showed tissue coagulative necrosis (yellow arrow), the cell morphology disappeared, and the germinal center tissue structure severely damaged (red arrow), but in other two groups only showed infiltration of inflammatory cells (black arrow) and porosity and disordered of cell arrangement (white arrow) (Fig. 7A). While the immunohistochemical changes on the lesions are qualitatively described in supplementary file (Table S13), all boars underwent a full pathological investigation and detailed immunohistochemical lesion scoring (Fig. 7B). In general, the lesions were assessed by an ordinal scale from 0 to 5 (health (0), minimal (1), mild (2), moderate (3), severe (4), critical (5)). Immunohistochemical scoring demonstrated that MGF100-deleted mutant is capable of providing better protection than MGF300-deleted mutant against tissue lesions. ASFV-Δ100 can induce more significant innate immune responses in vitro. The underlying mechanism of why ASFV-Δ100 is capable of conferring more protection against homologous challenge than ASFV-Δ300 is further explored. In the present study, ASFV-Δ100 is capable of triggering more Differentialiy expressed genes (DEGs) at 18 hpi and 30 hpi when compared with ASFV-WT virus and ASFV-Δ300, irrespective of up-regulated DEGs and down-regulated DEGs (Fig. 8A). The hierarchical clustering of DEGs shows that ASFV-Δ100 is able to trigger a higher level of interferon regulatory and inflammatory genes, as exemplified by CXCL10, PLAC8, DDX60, ISG20, IFIT family and TMEM156 when compared to ASFV-WT virus (Fig. 8B). However, ASFV-Δ300 infection weakened its induction capability of antiviral response as compared to the ASFV-WT virus, as evidenced by the fact that the levels of some innate antiviral genes were reduced. The results were also proved by GO-KEGG analyze, which showed that ASFV-Δ100 enriched more significant DEGs in antiviral and innate immune pathway, including defense response to virus, negative regulation of viral genome replication, type Ⅰ interferon signaling pathway and response to interferon-γ (Fig. 8C). How those interferon regulatory and inflammatory genes are involved in the induction of protection by ASFV-Δ100 are not clear, but we could primarily conclude that induction of innate antiviral response and interferon signaling within the host innate immune system is a key factor which contribute to the potential of ASFV-Δ100 as a promising safer and effective live attenuated ASFV vaccine candidate. Discussion African swine fever virus is notorious for causing great economic loss and being a threat to pig industry in every country it is prevalent. Traditionally been present in the African continent since 2005, it first leapt into east Europe in 2007 when the disease was confirmed in the Caucasus region of Georgia 9 . From there, the ASF sporadically occurred in the European Union (EU) affecting wild boars in particular. In 2018 the virus spread to China, which marked the first occurrence of ASF in Asia 10, 11 . Later ASF was observed to spread to Oceania in 2019/2020 and reappear in the Americas in 2021 12, 13 . Very recently from 2020 to 2024, ASF is further spreading in Africa, Asia and Europe. The wide-scale spread of ASF worldwide rationalizes the emergent need for vaccines. In search of reference we are informed that ASFV live attenuated vaccine is making more progress when compared to other types of vaccines. In particular, ASFV-G-ΔI177L, a modified-live vaccine that has been formulated into a commercially available vaccine, is gaining more and more attention. In 2020, it is reported that development of a highly effective live-attenuated ASFV vaccine by deletion of I177L gene leads to sterile immunity against the current highly virulent epidemic Eurasia strain 14 . Mechanically, I177L is reported to induce host inflammatory responses by facilitating the TRAF6-TAK1 axis and NLRP3 inflammasome assembly 15 . Later, a subset of live attenuated vaccines was developed based on the I177L gene-based deletion backbone and proved to be effective in pigs. Meanwhile, ASFV-G-ΔI177L could reach the similar and comparable safety and efficacy in challenge studies either by the oronasal (ON) route or by the intramuscular (IM) administration 16 . The ASFV-G-ΔI177L was first licensed and used in Vietnam with good feedback from the pig farms, though some concerns were raised with the deficit of less protection against the emerging recombinant Genotype Ⅰ/Ⅱ strain and genetical unstability in vitro and in vivo 17, 18 . Though live attenuated vaccine is under debate and controversary, ASFV-G-ΔI177L displays promising options for constraining ASF. Another live attenuated vaccine ASFV-G-ΔMGF was also licensed in Vietnam for commercial usage, although the follow-up research of this vaccine is rare. However, it proves that multiple gene-deletion of MGFs is a promising tool for ASFV attenuation and live attenuated vaccine development. However, the role of individual MGF gene is differential and not fully characterized, let alone the combinational effects of deleting members of ASF MGFs on virus attenuation, especially those from different families. In this study, we pioneered the complete deletion of MGF100 and MGF300 families from the Genotype Ⅱ Eurasian virulent strain on virus replication, virulence, and induction of protection. At the same time, we proved that compared to MGF300, MGF100 is acting as a more crucial virulence factor and a much stronger suppressor of host immune response. As a result, ASFV-Δ100 is more attenuated, as evidenced by no death and relatively rare clinical signs in the vaccination study and more protection in the challenge study, as exemplified by 80% protection and relatively light macroscopic findings. Those data imply that MGF100 is a promising target for developing live attenuated vaccine against ASFV. There are some limitations in our study. As we know, MGFs are not involved in virus assembly and not components of viral particles. However, in our study, we find that MGF100-2L deletion results in the appearance of untypical structure which is not often observed in the ASFV-WT virus-infected cells. In the preparation of virus infected cell samples, we harvested the cells for analysis following 48 h infection in a multistep growth cycle. In this regard, what stage the assembled viruses are at is not clear. As a result, whether MGF100-2L deletion affects the early stage of virus assembly or late stage of assembly is not clear, which will inspire us to go to more details in the future. Materials and Methods Cells, Viruses and reagents. Porcine bone marrow-derived macrophages (BMDMs) were isolated from femurs and tibias of 4-week-old crossbred piglets through aseptic dissection 19 . BMDMs and HEK293T cell lines were cultured with RPMI 1640 medium (Invitrogen, catalog no. C11875500BT), supplemented with 20% fetal bovine serum (Excell, catalog no. FSP500*5), 1% antibiotics (Solarbio, catalog no. P1410) and 10 ng/mL recombinant porcine granulocyte macrophage colony-stimulating factor (GM-CSF, ThermoFisher Scientific, catalog no. 14-7331-81). All the cells were maintained in cell culture plate (Corning ® 6/12/96-well plate, catalog no. 3516/3513/3599) in an incubator with 5% CO 2 at 37 ℃. The ASFV CN/GS/2018 strain (GCA_004135325.1) was used to construct the gene-deletion mutants and for the experiments involving in the parental wild-type ASFV (ASFV-WT). The VP72, VP30 and VP54 antibodies used in this experiment were produced by our laboratory and stored at -80 ℃. The DYKDDDDK tag (D6W5B) rabbit mAb (Binds to same epitope as Sigma’s Anti-FLAG ® M2 Antibody) (catalog no. 14793S), HA-tag (6E2) mouse mAb (catalog no. 2367S), Anti-mouse IgG(H+L), F(ab’) 2 fragment (Alexa Fluor ® 488 Conjugate) (catalog no. 4408S), Anti-rabbit IgG (H+L), F(ab') 2 Fragment (Alexa Fluor ® 594 Conjugate) (catalog no. 8889S) and DAPI (catalog no. 4083S) were purchased from CST. Anti-β-Actin mouse monoclonal antibody (1C7) was purchased from Abbkine (catalog no. A01010). HRP-conjugated goat anti-rabbit IgG (catalog no. BF03008) and goat anti-mouse IgG (catalog no. BF03001) were purchased from BIODRAGON. Construction of MGF-deleted ASFV mutants Schematic diagram depicting the deletion of MGF100 and MGF300 families was shown in Figure 3A and Figure 4A, respectively. In the case of MGF100 family, as MGF100-1R and MGF100-2L/3L are distributed at different genomic regions, ASFV-Δ100 is constructed by replacing MGF100-1R with the reporter gene EGFP under the control of ASFV VP72 promoter as well as replacing MGF100-2L/3L with VP72-mCherry reporter cassette. For MGF300 family, as all the members cluster at the same region, ASFV-Δ300 is constructed by the deletion of MGF300 family and insertion of the VP72-mCherry reporter cassette. The resulting virus from each homologous recombination event was purified by first isolating sing cells expressing the expected fluorescent proteins via fluorescence-activated cell sorting (FACS) and later seeding them into fresh BMDMs. All the recombinant viruses undergo at least 10 rounds of single cell sorting combined with limiting dilutions. The virus obtained from the last round of purification procedure was proliferated in BMDMs to generate virus stocks. To ensure the absence of parental ASFV CN/GS/2018 and the desired complete deletions of targeted genes in each recombinant mutant genome, virus DNA was extracted and subject to PCR as well as whole genome sequencing for further purity confirmation. The protocol was described previously with minor modifications 5, 20-22 . Next-generation sequencing of ASFV genomes. BMDMs were seeded in 96-well plates and infected with ASFV-Δ100 and ASFV-Δ300 respectively (MOI = 1). At 72 hpi ASFV genome was extracted from the cell cultures following the protocol by E.Z.N.A. ® Tissue DNA Kit (OMEGA, catalog no. D3396-02). Full-length whole genome sequencing was performed using the MGI SEQ 2000 platform and annotated based on NCBI NT (de redundant nucleotide Library) and NR (de redundant amino acid Library) databases. Sequencing depth and genome coverage were systematically analyzed using the MGI SEQ 2000 platform. The results were defined as reliable when target regions achieved a mean depth of 30,000X, as shown in Figure S3. Virus titration The wild-type ASFV CN/GS/2018 and each mutant virus was quantified by using the hemadsorption (HAD) assay as described previously with minor modifications 23 . BMDMs were seeded in 96-well plates. The virus suspensions were 10 times serially diluted and seeded to the plates. The HAD results were recorded on day 5 post-inoculation (p.i.), and 50% HAD doses (HAD 50 ) were calculated by using the method of Reed and Muench 24 . Growth kinetics of ASFV in BMD Ms BMDMs were grown in 96-well cell culture plates and infected with either parental strains (ASFV CN/GS/2018) or gene-deleted mutant strains (ASFV-Δ100 and ASFV-Δ300) at an MOI of 0.1. The samples were harvested at 2, 12, 24, 36, 48, 72 and 96 hours postinfection (hpi) and titrated by HAD assay. The results were depicted as multi-step growth curve. Biosafety statement and facility All experiments with live ASFVs were carried out within the biosafety level 3 (BSL-3) facilities in Lanzhou Veterinary Research Institute (LVRI), CAAS approved by the Ministry of Agriculture and Rural Affairs and China National Accreditation Service for Conformity Assessment. Animal study Fourteen ASFV-free Large White outbred pigs typically weighing about 30 kg were obtained from a licensed livestock farm and were randomly divided into two groups housed in two separated rooms in Biosafety Level 3 laboratory (BSL-3) in Lanzhou Veterinary Research Institute (LVRI), CAAS. One group (ear tag No. 5400, 5401, 5410, 5415, 5418, 5422 and 5437) were inoculated intramuscularly (IM) with ASFV-Δ100 (10 6.0 HAD 50 /pig) referring to as ASFV-Δ100 group, whereas the other group (ear tag No. 5412, 5414, 5426, 5427, 5432, 5433 and 5386) were administered with the same dose of ASFV-Δ300 referring to as ASFV-Δ300 group. Following 21 days vaccination, 2 out of 7 pigs died in ASFV-Δ300 group while all the 7 pigs survived in ASFV-Δ100 group. In the lethal challenge experiment, all 5 residual pigs in ASFV-Δ300 group were challenged IM with a lethal dose (10 2 .0 HAD 50 ) of highly virulent ASFV CN/GS/2018 strain. In order to compare the protective rate in two groups, 5 out of 7 pigs from ASFV-Δ100 group were randomly selected for lethal challenge experiment. Daily monitoring was conducted for each pig, which included recording rectal temperatures and observing the clinical signs of each pig, such as lethargy, anorexia, depression, vomiting, fever, skin hemorrhages, bloody diarrhea, and joint swelling. In addition, serum was collected every 2 days 25 , as were oral swabs, nasal swabs, and fecal swab samples. From each necropsied pig, tissues and organs (heart, liver, spleen, lung, kidney, submandibular lymph nodes, hepatogastric lymph nodes and mesenteric lymph nodes) were collected for viral load detection using qPCR and immuno-histochemistry examination. Anesthesia Procedure At the scientific or moderate severity humane endpoint, as defined in the project license, or at the end of animal experimentation, pigs were properly restrained using a snatch. Intramuscular administration of an overdose of Zoletil ® 50 (Virbac, catalog no. 50) was performed at 16 mg per kilogram of body weight. Monitoring of anesthesia depth is performed every 5-10 min. Once the animals demonstrated muscle relaxation, absence of movements, absence of palpebral and loss of consciousness 26 , they are subjected to euthanasia. All anesthesia and euthanasia procedures in this study were in compliance with animal welfare requirements and were strictly conducted in accordance with the "Standard Operating Procedures for Anesthesia and Euthanasia of Laboratory Animals" (Document No.: LVRI/HL/SY024-03-03), issued by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Laser confocal microscopy. For the analysis of subcellular localization of MGF100 and MGF300 proteins, HEK293T cells seeded on glass bottom cell culture dishes (NEST, catalog no. 801001) were transfected with overexpression plasmids or backbone vector for 30 h. Then, the cells were incubated with the relevant antibodies and examined with a Zeiss LSM 880 laser-scanning confocal microscope as described previously 27 . Western blotting Cultured cells were lysed in loading buffer containing 1 × SDS, heated at 100 ℃ for 10 min, then loaded onto SDS-PAGE gel. Then proteins were further electrophoretically transferred onto the polyvinylidene difluoride (PVDF) membrane (Merck-Millipore, catalog no. ISEQ00010) for 2 h with an electric current of 250 mA. Subsequently, the membrane was blocked with a mixture of 5 ml TBS blocking buffer (5% skimmed milk) for 1 h, followed by overnight incubation with primary antibodies at 4 ℃. The membrane was washed three times and then incubated with appropriate secondary antibody for 1 h. After washing three times, proteins were detected with Enhanced Chemiluminescent (NCE biotech, catalog no. P10300) 28 . Transmission electron microscopy For transmission electron microscopy (TEM) analysis of virus morphogenesis, BMDM cells were infected with different mutant strains for 30 h and fixed with 2.5% glutaraldehyde (Solarbio, catalog no. P1126) in 0.1 M phosphate buffer for 1 h at RT. Cells were then post-fixed with 2% osmium tetroxide and embedded in epoxy according to standard procedures. After polymerization, about 80 nm-thick sections were obtained and stained with uranyl acetate and lead citrate as previously described 29 . Samples were observed under the JEM-1400 transmission electron microscope operated at 80 kV. qPCR assay ASFV DNA was extracted from BMDMs or organ tissues by using E.Z.N.A. ® Tissue DNA Kit (OMEGA, catalog no. D3396-02) according to the manufacture’s protocols. The qPCR was carried out on a QuantStudio system (Applied Biosystems, USA) by using Pro taq HS premix probe qPCR kit (ACCURATE BIOLOGY AG, catalog no. AG11704). RNA-seq analysis BMDM cells were mock infected or infected with WT ASFV, ASFV-Δ100 and ASFV-Δ300 mutants for indicated time periods (18 and 30 hpi). Cells were harvested for RNA extraction using SteadyPure Universal RNA Extraction Kit (ACCURATE BIOLOGY AG, catalog no. AG21017). The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The original results were analyzed on the cloud platform of Oebiotech (https://cloud.oebiotech.com/#/home). The P values were adjusted using the Benjamini-Hochberg method. A corrected P value of 2 were set as the thresholds for significantly differential gene expression. Statistic al analysis Data are presented as Mean ± SEM. Comparisons between groups were performed with Mann-Whitney test using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered significant at a p value less than 0.05. Declarations Compliance and ethics The authors declare that they have no conflict of interest. All animal experiments related to ASFV were conducted in compliance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology (China). The protocols were approved by the Committee on Animal Research and Ethics of Lanzhou Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Sciences (CAAS) and Ethics Committee for Animal Experimentation of Gansu Province, China. Acknowledgments This work was supported by the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2024KJ14), the National Key R&D Program of China (2021YFD1800100), the Fundamental Research Funds for the Central Universities, China Agriculture Research System of Ministry of Finance and Ministry of Agriculture and Rural Affairs (CARS-35), Project of National Center of Technology Innovation for Pigs (NCTIP-XD/C03), Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSLPDCP-202302 and CAAS-ASTIP-2024-LVRI), Science and Technology Program of Gansu Province (24JRRA01), the China Postdoctoral Science Foundation (2023M733817) and the Postdoctoral Fellowship Program (Grade B) of China Postdoctoral Science Foundation (GZB20230857). Author contributions W.D., F.X. and H.Z. put forward the concept; F.X. and W.D. wrote the original manuscript text; W.D. reviewed and edited; F.X., T.L. and Y.D. prepared figures 1-3; F.X., M.D., H.L., Z.S., H.T. and J.H. prepared figures 4-7; F.X. and W.D. prepared figure 8; H.Z., M.D. and F.X. acquired fundings. All authors reviewed the manuscript. References Spinard, E.et al. Full genome sequence for the African swine fever virus outbreak in the Dominican Republic in 1980. Scientific reports 13 , 1024 (2023). Wang, N.et al. Architecture of African swine fever virus and implications for viral assembly. Science 366 , 640-644 (2019). Tao, D.et al. One year of African swine fever outbreak in China. Acta Trop 211 , 105602 (2020). Zhu, Z.et al. Classification and characterization of multigene family proteins of African swine fever viruses. Brief Bioinform 22 , (2021). O'donnell, V.et al. African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus. J Virol 89 , 6048-56 (2015). Reis, A. L.et al. Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response. Vaccine 34 , 4698-4705 (2016). Fan, W.et al. Synergistic effect of the responses of different tissues against African swine fever virus. Transbound Emerg Dis 69 , e204-e215 (2022). Sehl-Ewert, J.et al. Pathology of African Swine Fever in Reproductive Organs of Mature Breeding Boars. Viruses 15 , (2023). Njau, E. P.et al. The first genotype II African swine fever virus isolated in Africa provides insight into the current Eurasian pandemic. Scientific reports 11 , 13081 (2021). Wen, X.et al. Genome sequences derived from pig and dried blood pig feed samples provide important insights into the transmission of African swine fever virus in China in 2018. Emerg Microbes Infect 8 , 303-306 (2019). Zhao, D.et al. Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg Microbes Infect 8 , 438-447 (2019). Ito, S., Bosch, J., Martínez-Avilés, M., and Sánchez-Vizcaíno, J. M. The Evolution of African Swine Fever in China: A Global Threat? Front Vet Sci 9 , 828498 (2022). Ruiz-Saenz, J.et al. African swine fever virus: A re-emerging threat to the swine industry and food security in the Americas. Frontiers in microbiology 13 , 1011891 (2022). Borca, M. V.et al. Development of a Highly Effective African Swine Fever Virus Vaccine by Deletion of the I177L Gene Results in Sterile Immunity against the Current Epidemic Eurasia Strain. J Virol 94 , (2020). Wu, P. X.et al. African swine fever virus I177L induces host inflammatory responses by facilitating the TRAF6-TAK1 axis and NLRP3 inflammasome assembly. J Virol 99 , e0208024 (2025). Borca, M. V.et al. ASFV-G-∆I177L as an Effective Oral Nasal Vaccine against the Eurasia Strain of Africa Swine Fever. Viruses 13 , (2021). Van Den Born, E.et al. African swine fever virus vaccine strain Asfv-G-∆I177l reverts to virulence and negatively affects reproductive performance. Npj Vaccines 10 , 46 (2025). Diep, N. V.et al. Genotype II Live-Attenuated ASFV Vaccine Strains Unable to Completely Protect Pigs against the Emerging Recombinant ASFV Genotype I/II Strain in Vietnam. Vaccines (Basel) 12 , (2024). Gao, J. Y.et al. A new and efficient culture method for porcine bone marrow-derived M1-and M2-polarized macrophages. Vet Immunol Immunop 200 , 7-15 (2018). Rathakrishnan, A.et al. Deletion of the K145R and DP148R Genes from the Virulent ASFV Georgia 2007/1 Isolate Delays the Onset, but Does Not Reduce Severity, of Clinical Signs in Infected Pigs. Viruses 13 , (2021). O'donnell, V.et al. African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge. J Virol 89 , 8556-66 (2015). Monteagudo, P. L.et al. BA71ΔCD2: a New Recombinant Live Attenuated African Swine Fever Virus with Cross-Protective Capabilities. J Virol 91 , (2017). Malmquist, W. A. and Hay, D. Hemadsorption and cytopathic effect produced by African Swine Fever virus in swine bone marrow and buffy coat cultures. American journal of veterinary research 21 , 104-8 (1960). Németh, B.et al. A systematic review of health economic models and utility estimation methods in schizophrenia. Expert review of pharmacoeconomics & outcomes research 18 , 267-275 (2018). Weesendorp, E., Stegeman, A., and Loeffen, W. Dynamics of virus excretion via different routes in pigs experimentally infected with classical swine fever virus strains of high, moderate or low virulence. Vet Microbiol 133 , 9-22 (2009). Costea, R., Ene, I., and Pavel, R. Pig Sedation and Anesthesia for Medical Research. Animals-Basel 13 , (2023). Li, L. F.et al. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity. Journal of Virology 90 , 4412-4426 (2016). Xu, F.et al. IFIT3 mediated the type I interferon antiviral response by targeting Senecavirus A entry, assembly and release pathways. Vet Microbiol 275 , 109594 (2022). Matamoros, T.et al. African Swine Fever Virus Protein pE199L Mediates Virus Entry by Enabling Membrane Fusion and Core Penetration. Mbio 11 , (2020). Additional Declarations No competing interests reported. Supplementary Files SupplementFiles.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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6859140","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":476267395,"identity":"ebf39937-ac36-471f-ad7e-67ff926c7195","order_by":0,"name":"Wen Dang","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Dang","suffix":""},{"id":476267396,"identity":"b1547131-b60a-418f-90eb-f54ec8e271d5","order_by":1,"name":"Fan Xu","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Xu","suffix":""},{"id":476267399,"identity":"1858faef-b4b8-49eb-9940-e6e9adca07b8","order_by":2,"name":"Mingyang Ding","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Mingyang","middleName":"","lastName":"Ding","suffix":""},{"id":476267400,"identity":"042d9a8a-bf54-47af-b499-c70087b153c7","order_by":3,"name":"Tao Li","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Li","suffix":""},{"id":476267401,"identity":"38cb3195-bdbe-4525-bc1d-8a3b1dee1226","order_by":4,"name":"Yu Du","email":"","orcid":"","institution":"Lanzhou University, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Du","suffix":""},{"id":476267402,"identity":"01019070-03c9-44ec-9c90-f506ab9bb130","order_by":5,"name":"Huanan Liu","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Huanan","middleName":"","lastName":"Liu","suffix":""},{"id":476267403,"identity":"aab6d767-ca7c-4ae9-9f2d-a1054dd987ab","order_by":6,"name":"Zhengwang Shi","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Zhengwang","middleName":"","lastName":"Shi","suffix":""},{"id":476267405,"identity":"56acdbe6-df96-44a2-8581-d8eeff7f9c33","order_by":7,"name":"Hong Tian","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Tian","suffix":""},{"id":476267408,"identity":"7713a0bd-d20f-460d-8118-776f3fb11050","order_by":8,"name":"Jijun He","email":"","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Jijun","middleName":"","lastName":"He","suffix":""},{"id":476267409,"identity":"581c3915-6b24-4268-8971-14b9b3dfe495","order_by":9,"name":"Haixue Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYDACCSBmbGDgAVEPEipqSNPCbPDgzDHitYAAm+TDFmbCOuRnNx97+HWHnYzBjeRjFYkNbAz87d0JeLUwzjmWbix7JplHckZa2o3EHTIMEmfObsCrhVkix0xaso2Zhx/IuJF4ho3BQCIXvxY2ifxvQC31PCBGQWIbM2EtPBI5bJIf2w6DbGFjIEqLhESamTRj23EeyZ5nxhIJZ47xEPSL/IzkZ5I/26rtDY4nP/z4o6JGjr+9F78WEGDmQXYpQeUgwPiDKGWjYBSMglEwYgEAiidDDMvSmrUAAAAASUVORK5CYII=","orcid":"","institution":"Lanzhou Veterinary Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Haixue","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-06-10 05:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6859140/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6859140/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85795746,"identity":"21d9b8d4-1976-4933-89ed-abf0964a4b25","added_by":"auto","created_at":"2025-07-01 19:31:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7110166,"visible":true,"origin":"","legend":"\u003cp\u003eThe biological characteristics of the MGF100 and MGF300 families.\u003c/p\u003e\n\u003cp\u003eTranscriptional dynamics of (A) MGF100 and (C) MGF300 members. Average cycle threshold values of the ASFV CN/GS/2018-infected BMDMs (MOI of 1) at 0, 3, 6, 9, 12, 15 and 18 hpi were examined by qRT-PCR. Instead, CP204L and B646L were used as early and late genes, respectively. Intracellular localization of (B) MGF100 and (D) MGF300 proteins. HEK293T cells were transfected with plasmids pFlag-MGF100-1R, pFlag-MGF100-2L, pFlag-MGF100-3L, pFlag-MGF300-1L, pFlag-MGF300-2R or pFlag-MGF300-4L (2 μg/plasmid). After 36 hours, the cells were fixed, probed with the Flag rabbit RAb and detected by fluorescence microscope (Leica, DM16000B). Bars, 2 μm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/2bc413863f73dbb4765cddf2.png"},{"id":85795747,"identity":"16242f86-c709-48c9-a2d8-59d530ba02d7","added_by":"auto","created_at":"2025-07-01 19:31:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7033581,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction and characterization of ASFV-Δ100 and ASFV-Δ300 mutants.\u003c/p\u003e\n\u003cp\u003eSchematic representation of the gene(s) and region(s) deleted in (A) MGF100 and (B) MGF300 gene-deleted ASFV mutants. The numbers above the genome indicate the nucleotide position of targeted gene relative to the genome of ASFV CN/GS/2018. The deleted gene segments were replaced with the fluorescent reporters driven by the VP72 promotor. As a result, the cells infected with the corresponding reporter-labelled mutant virus were observed with Fluorescent Microscopy. To primarily prove the complete deletion of MGF100 and MGF300 members, six pairs of specific primers targeting the (A) MGF100-1R, MGF100-2L, MGF100-3L, (B) MGF300-1L, MGF300-2R and MGF300-4L were used for traditional PCR (lane 2) marker. Deionized water is used as negative control (lane 3) and ASFV-WT genome is loaded as positive control (lane 4). \u0026nbsp;(C) \u003cem\u003eIn vitro \u003c/em\u003ereplication characteristics of ASFV-Δ100 or ASFV-WT. BMDMs were infected at an MOI of 0.1 with ASFV-Δ100 or ASFV-WT. The cell cultures were collected at each time point, and growth curves were constructed by using the titer of each suspension at each time point. The data represent the results of three independent experiments. Error bars denote standard errors of the means. The significance of differences between groups (n = 3) was determined using Multiple t-test (*, P \u0026lt; 0.5; **, P \u0026lt; 0.01; ***, P \u0026lt; 0.001). (D) The replication kinetics were further determined by Western blot. (E) \u003cem\u003eIn vitro\u003c/em\u003e replication characteristics of ASFV-Δ300 or ASFV-WT. (F) The replication kinetics of MGF300 was further assessed by Western blot. The result is representative of three independent results.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/ed3721dabb64394dd54d6375.png"},{"id":85795751,"identity":"88f61956-94f6-4ecf-a2a9-815efc26c428","added_by":"auto","created_at":"2025-07-01 19:31:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22101103,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of cells infected with different mutants.\u003c/p\u003e\n\u003cp\u003e(A) and (B) BMDM cells infected with different mutants were harvested at 30 hpi and observed by TEM. Black arrowindicates mature virions; white arrow indicates immature virions; red arrowindicates deformed virions.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/8ec42fb324ed7124e9c25ce9.png"},{"id":85796190,"identity":"d3844e64-13d7-4c8c-8f7a-09f005d66af5","added_by":"auto","created_at":"2025-07-01 19:39:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7082027,"visible":true,"origin":"","legend":"\u003cp\u003eMGF100 and MGF300 were associated with the virulence of ASFV.\u003c/p\u003e\n\u003cp\u003e(A) Animal experiment design. The ASFV-free domestic pigs were first inoculated intramuscularly (IM) with ASFV-Δ100 or ASFV-Δ300 at 10\u003csup\u003e6.0\u003c/sup\u003e HAD\u003csub\u003e50\u003c/sub\u003e/pig, and then challenged with a lethal dose (10\u003csup\u003e2.0\u003c/sup\u003e HAD\u003csub\u003e50\u003c/sub\u003e/pig) of ASFV CN/GS/2018. Pigs were monitored for 18 days after challenge and then euthanized for pathological evaluation. During the period of experiment, rectal temperature was detected the other day, the same for collection of blood and swabs. At the end of experiment, surviving pigs were euthanized and indicated organs were collected for further analysis. The survival rates at the (B) vaccination period and (C) challenge period of two groups were demonstrated. The rectal temperatures of (D and E) MGF100 group and (F and G) MGF300 group were recorded every other day.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/dba6a200c2e2e6bba2de578a.png"},{"id":85795744,"identity":"4d5ba5d6-6f91-40df-8a65-558b2dd4b94e","added_by":"auto","created_at":"2025-07-01 19:31:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6166802,"visible":true,"origin":"","legend":"\u003cp\u003eBoth ASFV-Δ100 and ASFV-Δ300 induce protection and antibody response against lethal homologous challenge.\u003c/p\u003e\n\u003cp\u003eASFV genomic copies in the whole blood of the pigs from ASFV-WT group (B and D), ASFV-Δ100 (A and B) and ASFV-Δ300 (C and D) were detected by qPCR. The VP30levels in the serum collected from pigs in the vaccination period (E and G) and challenge period (F and H). The results were shown as blocking percentages. Immunoconversion is defined as positive when the blocking rate for anti-VP30 antibodies is above 50%. Group ASFV-Δ100 is represented in red, group ASFV-Δ300 is represented in green and group ASFV-WT is represented in black.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/c1551639a92188835ab27e5e.png"},{"id":85795748,"identity":"e6f2402b-3ad1-4466-9a40-69bcdf30b6b4","added_by":"auto","created_at":"2025-07-01 19:31:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12697008,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between viral loads and postmortem lesions.\u003c/p\u003e\n\u003cp\u003e(A) Virus titers in tissues from 2 groups of challenged pigs (red, ASFV-Δ100, n = 7; green, ASFV-Δ300, n = 7) and control pigs (black, ASFV-WT, n = 5). A total of 20 mg of tissue samples were homogenized, vortexed, clarified, and subjected to copy number detection. (*, p \u0026lt; 0.05). (B) Comparative postmortem lesions. The images show representative organs from pigs in each group (ASFV-WT: No. 5542; ASFV-Δ100: No. 5415 and ASFV-Δ300: No. 5427) as follows: ①heart；②liver；③spleen；④lung；⑤kidney；⑥submandibular lymph node；⑦hepatogastric lymph node and ⑧mesenteric lymph node. The hemorrhage and lesions were marked by red arrow.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/334b03a91bd15f3a6571c3f1.png"},{"id":85795753,"identity":"392f2938-3892-4f48-8c4a-c1d6a8bc43f3","added_by":"auto","created_at":"2025-07-01 19:31:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":49160588,"visible":true,"origin":"","legend":"\u003cp\u003eScoring and characterization of histopathological lesions.\u003c/p\u003e\n\u003cp\u003e(A) Representative histopathological lesions in different tissue samples of pigs in each group (ASFV-WT: No. 5542; ASFV-Δ100: No. 5415 and ASFV-Δ300: No. 5427). Different colored arrows represent different pathological changes. Black arrow indicates neutrophil infiltration. Green arrow indicatesacute and diffuse hemorrhages. Red arrow indicates that the structure of germinal center disappeared and the structure was destroyed. Blue arrowindicates thatfibrous tissue proliferated and underwent substantial changes. Yellow arrowindicates that the cell tissue was coagulative necrotic and the cell morphology disappeared. White arrow indicatesthe germinal center cells were arranged loosely and disorderly. (B) Scores of histopathological lesions. The histopathological lesions of each pig were scored based on our self-developed reference standard for clinical presentations of respective organs.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/6df1bc937d41eda3b1a3bc9e.png"},{"id":85795750,"identity":"55ef9f89-b431-4fec-8969-8e123106653c","added_by":"auto","created_at":"2025-07-01 19:31:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3019768,"visible":true,"origin":"","legend":"\u003cp\u003eASFV-Δ100 induced a more pronounced innate immune response \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eBMDM were mocked infected or infected with ASFV-WT, ASFV-Δ100 and ASFV-Δ300 at an MOI of 0.1. Following the indicated infection time (18 and 30 hpi), cell cultures were subject to RNA-seq analysis. (A) The number of up- or down-regulated DEGs produced by cells stimulated with different mutants at different infection times compared to MOCK (P \u0026lt; 0.001, |fold change| ≥ 2) is described. (B) Hierarchical clustering of the DEGs identified in ASFV-infected BMDM. A total of 84 genes implicated in the innate immune response were probed over time and are displayed in a heat map. Each panel represents a particular gene, and the color depicts the fold change (FC) at the indicated time points. (C) Bubble histogram of significantly enriched GO-KEGG classifications of upregulated DEGs. The top 10 upregulated GO Terms involved in the three main categories, namely, BP-biological process, CC-cellular component and MF-molecular function, are ranked based on the counts of upregulated DEGs in ASFV-Δ100 or ASFV-Δ300-infected BMDMs compared to ASFV-WT infected samples at 18 or 30 hpi. The Y axis in the figure corresponds to the name of the pathway, the X axis corresponds to the enrichment score. The dots on the bubble chart represent the number of differential genes. The larger the dot, the more up-regulated DEGs in a specific pathway. The color depicts the p-value at the indicated pathway.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/855ff5612070f20381dc2450.png"},{"id":93109780,"identity":"b7420a3d-5715-41c2-9980-e229c9ad0fcc","added_by":"auto","created_at":"2025-10-09 07:33:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":107064819,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/d952a989-c801-4ac7-a055-746a4cc214b4.pdf"},{"id":85795752,"identity":"6644cc5e-6782-473c-83a5-d4ba3a2810a1","added_by":"auto","created_at":"2025-07-01 19:31:21","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5265591,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementFiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-6859140/v1/a6f73944b196b9d669aa9403.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"MGF100 but not MGF300 family is a potential multigene-deleted target for ASFV attenuation and live attenuated vaccine development","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAfrican swine fever (ASF) is a highly infectious and severe haemorrhagic viral disease of swine with high mortality. The disease is circulating on the African, European and Asian continents, as well as recently in Dominican Republic and Haiti \u003csup\u003e1\u003c/sup\u003e. As the only member of the \u003cem\u003eAsfarviridae\u003c/em\u003e family, genus \u003cem\u003eAsfivirus\u003c/em\u003e, the etiological agent ASF virus (ASFV) is a double stranded DNA virus wrapped in a multi-layer icosahedral structure with a diameter of about 200 nm and notably the only DNA virus (arbovirus) that can be transmitted through arthropods \u003csup\u003e2\u003c/sup\u003e. The spread of the virus continues, exacerbated by the fact that nowadays there are no effective therapeutic options against ASFV infection \u003csup\u003e3\u003c/sup\u003e. Vaccination is seemingly a promising tool, of which the live attenuated vaccines so far make the most progress in term of efficacy and protection, although some concerns are raised about the safety issues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe multigene families (MGFs) are mainly distributed at both ends of the viral genome and play important roles in affecting viral virulence, regulating interferon response and inflammatory response. MGFs are divided into five major categories, namely MGF100, MGF110, MGF300, MGF360 and MGF505 \u003csup\u003e4\u003c/sup\u003e. MGFs have always been good options for deletion as a way of virus attenuation, in which combinational deletions of MGF360 and MGF505 genes are well characterized. The virulent Genotype\u0026nbsp;Ⅱ\u0026nbsp;Georgia 2007/1 isolate harboring combinational deletions of MGF360 and MGF505 genes is highly attenuated in swine and is able to provide protection against homologous challenge \u003csup\u003e5\u003c/sup\u003e. In parallel, the same deletions exerted same effects on Genotype Ⅰ virulent strain, as deletions of MGF360 genes (MGF360-10L, 11L, 12L, 13L and 14L) and MGF505 genes (MGF505-1R, 2R and 3R) and interrupting genes (MGF360-9L and MGF505-4R) in the genome of Benin 97/1 reduced its virulence in domestic pigs and induced a protective response \u003csup\u003e6\u003c/sup\u003e. Those data imply that multiple-gene deletions, especially deletions of MGFs are a promising way to produce an attenuation when there is no clear identification of the function and role of individual target genes (Table S12).\u003c/p\u003e\n\u003cp\u003eCompared to the MGF110, MGF360 and MGF505 families, the MGF100 and MGF300 families are relatively less studied. So far, the role of the individual genes from MGF100 and MGF300 families is poorly understand. It is empirically suggested that compared to single-gene deletions, multiple-gene deletions are more capable of attenuating ASFV and developing live attenuated candidate. In this regard, two ASFV candidates were developed based on the highly virulent Genotype\u0026nbsp;Ⅱ\u0026nbsp;Eurasian strain\u0026nbsp;ASFV\u0026nbsp;CN/GS/2018\u0026nbsp;with one losing the MGF100 family and the other losing the MGF300 family, namely ASFV-Δ100 and ASFV-Δ300, respectively. Later the pathogenicity, immunogenicity and protective response of two candidates were compared in animal experiment. We find that\u0026nbsp;ASFV-Δ100\u0026nbsp;is able to find a better balance in virulence, immunogenicity and protective response, as\u0026nbsp;ASFV-Δ100\u0026nbsp;infection results in no or extremely slight ASFV-compatible clinical signs. On the contrary,\u0026nbsp;ASFV-Δ300\u0026nbsp;is not completely attenuated, as evidenced by the death of 2 pigs following vaccination. Meanwhile, irrespective of same protection rates in the residual pigs following lethal challenge, the\u0026nbsp;ASFV-Δ300\u0026nbsp;group demonstrated more severe post-mortem signs and immuno-histochemistry injury when compared to\u0026nbsp;ASFV-ΔMGF100 group.\u0026nbsp;At the same time, we also\u0026nbsp;find\u0026nbsp;that the\u0026nbsp;virion morphogenesis\u0026nbsp;changes\u0026nbsp;significantly after deletion of MGF100-2R, showing\u0026nbsp;a subset of forms, including curved, linear and semilunar membranes, which may\u0026nbsp;partially explain the\u0026nbsp;decline of\u0026nbsp;virus\u0026nbsp;virulence.\u0026nbsp;RNA-sequencing data also display the fact that\u0026nbsp;ASFV-Δ100\u0026nbsp;is capable of inducing more robust innate immune compared to\u0026nbsp;ASFV-Δ300, which potentially uncovered its stronger protective capacity.\u0026nbsp;In summary, MGF100 but not MGF300 family is a\u0026nbsp;more promising\u0026nbsp;multigene-deleted target for\u0026nbsp;ASFV\u0026nbsp;attenuation and live attenuated vaccine development. The\u0026nbsp;ASFV-Δ100\u0026nbsp;is a promising live attenuated vaccine candidate, though further research on it is needed.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eConservation and transcription kinetics of MGF100 and MGF300 families.\u003c/h2\u003e\n\u003cp\u003eThe open reading frames (ORFs) of MGF100 family are located at positions 11094-11468 (MGF100-1R), 179519-179944 (MGF100-2L) and 180309-180617 (MGF100-3L) of the full-length genome sequence of ASFV CN/GS/2018. Likewise, four members of MGF300 family are located at positions 19732-20538 (MGF300-1L), 21365-21847 (MGF300-2R), 21937-22251 (MGF300-3L) and 21937-22929 (MGF300-4L). As MGF300-3L is a C-terminally truncated version of MGF300-4L, it is not further characterized in this study. A comparative study of conservation is performed to evaluate the conservation of MGF100 and MGF300 members across variants of ASFV Genotypes Ⅰ, Ⅱ, Ⅳ, Ⅴ (data resource: The African Swine Fever Virus Database).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe amino acid identity of MGF100-1R is absolutely high across Genotype\u0026nbsp;Ⅰ/Ⅱ\u0026nbsp;strains, ranging from\u0026nbsp;94% to\u0026nbsp;100% (Fig. S1A,\u0026nbsp;Table S1). Paralleling with the conservation results from MGF100-1R, MGF100-2L demonstrated the same trends. Of significant note, two\u0026nbsp;Genotype\u0026nbsp;Ⅱ\u0026nbsp;variants with truncated version of MGF100-2L were observed, with the size of 48 amino acids\u0026nbsp;in Ukraine_Kyiv_2014_QED21767.1 and 75 amino acids\u0026nbsp;in Ukraine_Kyiv_2014_QED21766.1. Also, a relatively high conservation is observed, ranging from 95% to 97% in\u0026nbsp;Genotype I strains and from 82% to 90% in other\u0026nbsp;genotypes (Fig. S1B,\u0026nbsp;Table S2). The situation in MGF100-3L is relatively different. Across most\u0026nbsp;Genotype\u0026nbsp;Ⅱ\u0026nbsp;strains, a length size of 102 amino acids is dominantly observed with high identity. However, a longer length of 146 amino acids is observed in three\u0026nbsp;Genotype\u0026nbsp;Ⅱ\u0026nbsp;strains, namely South_Africa_KNP_Pretorisuskop_1996, Malawi_Tengani_1962 and South_Africa_Warmbaths_1987. Surprisingly,\u0026nbsp;Genotype I strains all carry a 146 amino-acid MGF100-3L protein with an identity of 84%. The amino acid identity in\u0026nbsp;Genotype\u0026nbsp;Ⅳ,\u0026nbsp;Ⅶ,\u0026nbsp;Ⅸ\u0026nbsp;and\u0026nbsp;Ⅺ\u0026nbsp;strains is divergent, ranging from 75% to 97% (Fig. S1C,\u0026nbsp;Table S3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the context of MGF300-1L, the identity is high, ranging from 95% in Genotype I to 100% in Genotype\u0026nbsp;Ⅱ\u0026nbsp;(Fig. S2A,\u0026nbsp;Table S4), which is the case for\u0026nbsp;MGF300-2R\u0026nbsp;(Fig. S2B,\u0026nbsp;Table S5). \u0026nbsp;Of note, there are two dominant versions of MGF300-4L, one is 254 amino acid length and the other is 330 amino acid length. Irrespective of the length, MGF300-4L is highly conserved in\u0026nbsp;Genotype\u0026nbsp;Ⅰ/Ⅱ\u0026nbsp;strains but diverse in other\u0026nbsp;genotype strains\u0026nbsp;(Fig. S2C, Table S6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious study demonstrated the transcriptome profile of ASFV genes, highlighting distinctive patterns of individual gene expressions. Three members of MGF100 demonstrated the same transcriptional trends during ASFV infection. Like VP30, the expression level of MGF100 members peaked at the very early of 3 hours post infection (hpi), stayed at a plateau for the next 6 h and began to decline starting at 9 hpi (Fig.\u0026nbsp;1A). However, MGF300 members are divergent in transcriptional patterns. MGF300-2R and MGF300-4L demonstrated the same trends as MGF100. On the contrary, MGF300-1L is proved to be a late gene, as its expression continuously increased over time (Fig.\u0026nbsp;1C).\u003c/p\u003e\n\u003cp\u003eThe spatial localization of MGF100 and MGF300 in ASFV-infected cells is further characterized. By employing IFA in 293T cells, we found proteins encoded by MGF100 and MGF300 were dominantly localized in the cytoplasm, except for MGF300-1L, as it demonstrated a dominant distribution in the cytoplasm and a discrete distribution in the nuclear (Fig. 1B and D).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConstruction and characterization of ASFV-\u0026Delta;100 and ASFV-\u0026Delta;300 mutants.\u003c/h2\u003e\n\u003cp\u003eTo understand how MGF100 family is involved in ASFV replication capacity \u003cem\u003ein vitro\u003c/em\u003e and virulence \u003cem\u003ein vivo\u003c/em\u003e, the three-gene-deleted ASFV-\u0026Delta;100 mutant was constructed and further characterized (Fig. 2A). The precision of MGF100 family deletion was further validated by RT-PCR (Fig. 2A) and whole-genome sequencing (Fig. S1B). As anticipated, expected deletion was well achieved. Likewise, ASFV-\u0026Delta;300 mutant was developed with the same strategy and demonstrated high efficiency of expected genomic modifications, as analyzed by RT-PCR (Fig. 2B) and whole-genome sequencing (Fig. S1C). However, apart from expected deletions, during the process of homologous recombination, some unexpected genomic modifications were observed, as summarized in Table S7 for ASFV-\u0026Delta;100 and Table S8 for ASFV-\u0026Delta;300.\u003c/p\u003e\n\u003cp\u003eIn depicting multi-step virus growth curves of two mutants, we found that ASFV-\u0026Delta;100 and ASFV-\u0026Delta;300 demonstrated the same trends in replication capacity during the first burst period, starting from 2 to 12 hpi. However, over time ASFV-\u0026Delta;100 and ASFV-\u0026Delta;300 viral loads were significantly reduced by approximately 0.5 log compared with ASFV-WT depending on the time points considered (Fig. 2C and E), indicating that MGF100 and MGF300 families are determinants for virus growth \u003cem\u003ein vitro\u003c/em\u003e. The consistent results were also observed at the protein level (Fig. 2D and F).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eMGF100-2R is potentially involved in the assembly of ASFV particles.\u003c/h2\u003e\n\u003cp\u003eThe MGF family is proved to be multifunctional in roles ranging from suppressing the type\u0026nbsp;Ⅰ\u0026nbsp;interferon response to virulence in pigs. However, the role of MGFs\u0026nbsp;in involving in ASFV morphogenesis is poorly understood. Cells infected with ASFV-WT or MGF300-deleted ASFV clearly showed whole virions in either mature (Fig.\u0026nbsp;3A, black arrow) or immature (Fig. 3A, white arrow) stages surrounding membrane assembly intermediates. Particularly, an electron dense protein/DNA accumulation was observed in the center of the factory. Meanwhile, the partly formed icosahedral structures are commonly observed, with the capsid protein layer accumulating on the outer face and inner core shell proteins assembling on the inner face. However, in the cells infected with MGF100-deleted ASFV, strikingly, we found that the membrane assembly intermediates displayed\u0026nbsp;a subset of forms, including curved, linear and semilunar membranes as well as membrane fragments devoid of the dense capsid or core shell protein layers (Fig.\u0026nbsp;3A, red arrow). To elucidate which member of MGF100 family is involved in ASFV morphogenesis, individual genes of the MGF100 family are deleted from the ASFV-WT. Our data suggests that deletion of MGF100-2L itself results in the changes of ASFV morphogenesis (Fig.\u0026nbsp;3B, red arrow), implying that MGF100-2L is involved in the assembly of ASFV particles, even though the exact mechanism is unclear.\u003c/p\u003e\n\u003ch2\u003eEvaluation of ASFV mutants virulence and protective efficacy in pigs.\u003c/h2\u003e\n\u003cp\u003eTo evaluate the potential of MGF100 and MGF300 families as virulence factors whose mutants can be used as live attenuated vaccines, \u003cem\u003ein vivo\u003c/em\u003e evaluation of safety, immunogenicity and protective efficacy was performed. In detail, domestic pigs weighing about 30 kg were intramuscularly (IM) vaccinated with ASFV-\u0026Delta;100 (n = 7) or ASFV-\u0026Delta;300 (n = 7)) at a dose of 10\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e.0\u003c/sup\u003e HAD\u003csub\u003e50\u003c/sub\u003e at which\u0026nbsp;was\u0026nbsp;lethal for pigs in the case of ASFV-WT.\u0026nbsp;During\u0026nbsp;a period of 21\u0026nbsp;days\u0026nbsp;observation, a serial of clinical symptoms compatible with ASFV infection were monitored (Fig.\u0026nbsp;4A). All animals in the ASFV-\u0026Delta;100 group survived and remained healthy, only two of which (No.\u0026nbsp;5400 and No.\u0026nbsp;5410)\u0026nbsp;displayed\u0026nbsp;recurrent transient fever at the very first beginning of vaccination\u0026nbsp;(Fig. 4B and D). However, two animals (No.\u0026nbsp;5386 and No.\u0026nbsp;5433) in the ASFV-\u0026Delta;300 group started to develop fever at 4\u0026nbsp;days post vaccination (dpv)\u0026nbsp;and died on 13 and 16 dpv (Fig.\u0026nbsp;4B and F).\u0026nbsp;The residual pigs are asymptomatic, however, remained a relatively worse health status, as evidenced by less food take and depression.\u0026nbsp;Apparently, ASFV-\u0026Delta;300 is\u0026nbsp;partially\u0026nbsp;attenuated\u0026nbsp;and\u0026nbsp;retained\u0026nbsp;some\u0026nbsp;level of virulence in pigs when compared to ASFV-\u0026Delta;100.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the coming evaluation of immunogenicity and protective efficacy, vaccinated pigs were later challenged with a lethal dose (10\u003csup\u003e2.0\u003c/sup\u003e HAD\u003csub\u003e50\u003c/sub\u003e) of ASFV-WT via IM route. To facilitate comparison between 2 mutants, five out of seven surviving pigs in the ASFV-\u0026Delta;100 group were selected for the challenge experiment. Five na\u0026iuml;ve animals challenged with ASFV-WT virus started to develop fever at 4 days post challenge (dpc), peaked at 6 dpc and subsequently died within 9 dpc (Fig. 4C and E). On the contrary, 4 out of 5 pigs in ASFV-\u0026Delta;100 and\u0026nbsp;ASFV-\u0026Delta;300 group survived the homologous lethal challenge, with one died on 9 dpc for the former group and one died on 13 dpc for the latter group (Fig. 4C, E and G; Table S9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further observe the clinical presentations of animals during vaccination and challenge, virus shedding and viremia were further quantitatively assessed. Pigs in the ASFV-\u0026Delta;100 group demonstrated fluctuating but relatively high level of viremia in the beginning of vaccination. The viremia tends to drop to low level at the end of the vaccination (18 - 20 dpv) (Fig. 5A). In the ASFV-\u0026Delta;300 group, two dead pigs displayed high level of viremia, which was consistent with other clinical symptoms. For the surviving pigs, they tend to have comparable level of viremia as ASFV-\u0026Delta;100 group (Fig. 5C). The situation of virus shedding in fecal and oral-nasal swabs is the same for both mutants (Fig. S5). During challenge period, surviving pigs in both groups developed the transient fever between 2 and 6 dpc but quickly regained the healthy status. Consistently, surviving pigs from both groups displayed a low or undetectable level of viremia and virus shedding, suggestive of good protection (Fig. 5B and D; Fig. S5), even the two dead pigs were immunogenic to high level of ASFV-WT replication. Those data primarily imply that ASFV-\u0026Delta;300 is attenuated but more pathogenic when compared to ASFV-\u0026Delta;100.\u003c/p\u003e\n\u003ch2\u003eASFV-\u0026Delta;100 demonstrated higher immunogenicity against homologous challenge.\u003c/h2\u003e\n\u003cp\u003eThe underlying protective mechanism of live attenuated vaccine against ASFV infection is not fully unclear, however, it is strongly believed that antibody response is playing a key role. In the vaccination phase, ASFV-\u0026Delta;100 group reach the peak of VP30 antibody at 12 dpv at a fast and uniform pace (Fig. 5E). In the ASFV-\u0026Delta;300 group, the VP30 response is a little bit delayed and inconsistent among pigs, though all reached the plateau at the end of vaccination (Fig. 5G). At the period of challenge experiment, the VP30 response remained at the same plateau, except one dead animal from ASFV-\u0026Delta;100 group demonstrated a slight decline of VP30 (Fig. 5F). On the contrary, all the naive pigs died before VP30 response was full activated. Those data imply that VP30 response is a potential marker for measuring the protective response exerted by live attenuated vaccine.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eASFV-\u0026Delta;100 is capable of providing more pathological protection on organs following lethal challenge.\u003c/h2\u003e\n\u003cp\u003eAs the protection rate is the same for ASFV-\u0026Delta;100\u0026nbsp;and\u0026nbsp;ASFV-\u0026Delta;300 in the animal challenge experiment, we were further prompted to compare the protection efficacy of both mutants at the pathological level. When detecting the viral loads in organ samples, it is well observed that viral loads in ASFV-\u0026Delta;100\u0026nbsp;group is significantly lower when compared to the\u0026nbsp;ASFV-\u0026Delta;300 group and ASFV-WT group (Fig. 6A). Those findings primarily indicate that ASFV-\u0026Delta;100 but not ASFV-\u0026Delta;300\u0026nbsp;is providing more protection against homologous challenge. In search of more evidence to support our hypothesis, the postmortem data demonstrated pulmonary congestion, pneumonia with caseous necrosis (occasionally accompanied by local calcification), foaming of the trachea and bronchus, and severe alveolar and interstitial pulmonary oedema in dead pigs from control group (red arrow) \u003csup\u003e7\u003c/sup\u003e. Specifically, spleen is characterized with enlargement and friability accompanied by dark red or even black round edges (red arrow). Petechiation on the capsule of the kidney is obviously and frequently observed with severe ecchymoses (cortexes and pelves) (red arrow). Apart from that, hepatic congestion, gallbladder bleeding, lymph node enlargement and severe haemorrhage are also common in organs from dead pigs (Fig. 6B, red arrow). On the contrary, the presence of ASFV-compatible pathological lesions, was less observed in the ASFV-\u0026Delta;100\u0026nbsp;and\u0026nbsp;ASFV-\u0026Delta;300 pigs. It seems that the severity of pathological lesions in both groups is greatly but equally relieved. To gain more evidence, histopathological observation of organ injury was further performed. Consistent with pathological findings, notably, the most obvious immunohistochemical changes associated with ASFV infection are multifocal interstitial hemorrhages, apoptosis/necrosis of cells and presence of moderate amounts of positively labeled cells consistent with histiocytes \u003csup\u003e8\u003c/sup\u003e. Particularly in pathology, organs infected with ASFV-WT virus showed large-scale interstitial neutrophilic infiltration (black arrow) and hemorrhage (green arrow), which in two immunized groups were no bleeding. The lymph nodes in the WT group showed tissue coagulative necrosis (yellow arrow), the cell morphology disappeared, and the germinal center tissue structure severely damaged (red arrow), but in other two groups only showed infiltration of inflammatory cells (black arrow) and porosity and disordered of cell arrangement (white arrow) (Fig. 7A).\u003c/p\u003e\n\u003cp\u003eWhile the immunohistochemical changes on the lesions are qualitatively described in supplementary file (Table S13), all boars underwent a full pathological investigation and detailed immunohistochemical lesion scoring (Fig. 7B). In general, the lesions were assessed by an ordinal scale from 0 to 5 (health (0), minimal (1), mild (2), moderate (3), severe (4), critical (5)). Immunohistochemical scoring demonstrated that MGF100-deleted mutant is capable of providing better protection than MGF300-deleted mutant against tissue lesions.\u003c/p\u003e\n\u003ch2\u003eASFV-\u0026Delta;100 can induce more significant innate immune responses in vitro.\u003c/h2\u003e\n\u003cp\u003eThe underlying mechanism of why ASFV-\u0026Delta;100 is capable of conferring more protection against homologous challenge than ASFV-\u0026Delta;300 is further explored. In the present study, ASFV-\u0026Delta;100 is capable of triggering more Differentialiy expressed genes (DEGs) at 18 hpi and 30 hpi when compared with ASFV-WT virus and ASFV-\u0026Delta;300, irrespective of up-regulated DEGs and down-regulated DEGs (Fig. 8A). The hierarchical clustering of DEGs shows that ASFV-\u0026Delta;100 is able to trigger a higher level of interferon regulatory and inflammatory genes, as exemplified by CXCL10, PLAC8, DDX60, ISG20, IFIT family and TMEM156 when compared to ASFV-WT virus (Fig. 8B). However, ASFV-\u0026Delta;300 infection weakened its induction capability of antiviral response as compared to the ASFV-WT virus, as evidenced by the fact that the levels of some innate antiviral genes were reduced. The results were also proved by GO-KEGG analyze, which showed that ASFV-\u0026Delta;100 enriched more significant DEGs in antiviral and innate immune pathway, including defense response to virus, negative regulation of viral genome replication, type Ⅰ interferon signaling pathway and response to interferon-\u0026gamma; (Fig. 8C). How those interferon regulatory and inflammatory genes are involved in the induction of protection by ASFV-\u0026Delta;100 are not clear, but we could primarily conclude that induction of innate antiviral response and interferon signaling within the host innate immune system is a key factor which contribute to the potential of ASFV-\u0026Delta;100 as a promising safer and effective live attenuated ASFV vaccine candidate.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAfrican swine fever virus is notorious for causing great economic loss and being a threat to pig industry in every country it is prevalent. Traditionally been present in the African continent since 2005, it first leapt into east Europe in 2007 when the disease was confirmed in the Caucasus region of Georgia \u003csup\u003e9\u003c/sup\u003e. From there, the ASF sporadically occurred in the European Union (EU) affecting wild boars in particular. In 2018 the virus spread to China, which marked the first occurrence of ASF in Asia \u003csup\u003e10, 11\u003c/sup\u003e. Later ASF was observed to spread to Oceania in 2019/2020 and reappear in the Americas in 2021 \u003csup\u003e12, 13\u003c/sup\u003e. Very recently from 2020 to 2024, ASF is further spreading in Africa, Asia and Europe. The wide-scale spread of ASF worldwide rationalizes the emergent need for vaccines.\u003c/p\u003e\n\u003cp\u003eIn search of reference we are informed that ASFV live attenuated vaccine is making more progress when compared to other types of vaccines. In particular, ASFV-G-ΔI177L, a modified-live vaccine that has been formulated into a commercially available vaccine, is gaining more and more attention. In 2020, it is reported that development of a highly effective live-attenuated ASFV vaccine by deletion of I177L gene leads to sterile immunity against the current highly virulent epidemic Eurasia strain \u003csup\u003e14\u003c/sup\u003e. Mechanically, I177L is reported to induce host inflammatory responses by facilitating the TRAF6-TAK1 axis and NLRP3 inflammasome assembly \u003csup\u003e15\u003c/sup\u003e. Later, a subset of live attenuated vaccines was developed based on the I177L gene-based deletion backbone and proved to be effective in pigs. Meanwhile, ASFV-G-ΔI177L could reach the similar and comparable safety and efficacy in challenge studies either by the oronasal (ON) route or by the intramuscular (IM) administration \u003csup\u003e16\u003c/sup\u003e. The ASFV-G-ΔI177L was first licensed and used in Vietnam with good feedback from the pig farms, though some concerns were raised with the deficit of less protection against the emerging recombinant Genotype\u0026nbsp;Ⅰ/Ⅱ\u0026nbsp;strain and genetical unstability \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e17, 18\u003c/sup\u003e. Though live attenuated vaccine is under debate and controversary, ASFV-G-ΔI177L displays promising options for constraining ASF.\u003c/p\u003e\n\u003cp\u003eAnother live attenuated vaccine ASFV-G-ΔMGF was also licensed in Vietnam for commercial usage, although the follow-up research of this vaccine is rare. However, it proves that multiple gene-deletion of MGFs is a promising tool for ASFV attenuation and live attenuated vaccine development. However, the role of individual MGF gene is differential and not fully characterized, let alone the combinational effects of deleting members of ASF MGFs on virus attenuation, especially those from different families. In this study, we pioneered the complete deletion of MGF100 and MGF300 families from the Genotype\u0026nbsp;Ⅱ\u0026nbsp;Eurasian virulent strain on virus replication, virulence, and induction of protection. At the same time, we proved that compared to MGF300, MGF100 is acting as a more crucial virulence factor and a much stronger suppressor of host immune response. As a result, ASFV-Δ100 is more attenuated, as evidenced by no death and relatively rare clinical signs in the vaccination study and more protection\u0026nbsp;in the challenge study, as exemplified by\u0026nbsp;80% protection and relatively light macroscopic findings. Those data imply that MGF100 is a promising target for developing live attenuated vaccine against ASFV.\u003c/p\u003e\n\u003cp\u003eThere are some limitations in our study. As we know, MGFs are not involved in virus assembly and not components of viral particles. However, in our study, we find that MGF100-2L deletion results in the appearance of untypical structure which is not often observed in the ASFV-WT virus-infected cells. In the preparation of virus infected cell samples, we harvested the cells for analysis following 48 h infection in a multistep growth cycle. In this regard, what stage the assembled viruses are at is not clear. \u0026nbsp;As a result, whether MGF100-2L deletion affects the early stage of virus assembly or late stage of assembly is not clear, which will inspire us to go to more details in the future.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCells, Viruses and reagents.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePorcine bone marrow-derived macrophages (BMDMs) were isolated from femurs and tibias of 4-week-old crossbred piglets through aseptic dissection \u003csup\u003e19\u003c/sup\u003e. BMDMs and HEK293T cell lines were cultured with RPMI 1640 medium (Invitrogen, catalog no. C11875500BT), supplemented with 20% fetal bovine serum (Excell, catalog no. FSP500*5), 1% antibiotics (Solarbio, catalog no. P1410) and 10 ng/mL recombinant porcine granulocyte macrophage colony-stimulating factor (GM-CSF, ThermoFisher Scientific, catalog no. 14-7331-81). All the cells were maintained\u0026nbsp;in cell culture plate (Corning\u003csup\u003e®\u003c/sup\u003e 6/12/96-well plate, catalog no. 3516/3513/3599)\u0026nbsp;in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026nbsp;℃.\u003c/p\u003e\n\u003cp\u003eThe ASFV CN/GS/2018 strain (GCA_004135325.1) was used to construct the gene-deletion mutants and for the experiments involving in the parental wild-type ASFV (ASFV-WT).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe VP72, VP30 and VP54 antibodies used in this experiment were produced by our laboratory and stored at -80\u0026nbsp;℃. The\u0026nbsp;DYKDDDDK tag (D6W5B) rabbit mAb\u0026nbsp;(Binds to same epitope as Sigma’s Anti-FLAG\u003csup\u003e®\u003c/sup\u003e M2 Antibody)\u0026nbsp;(catalog no.\u0026nbsp;14793S), HA-tag\u0026nbsp;(6E2) mouse mAb\u0026nbsp;(catalog no.\u0026nbsp;2367S),\u0026nbsp;Anti-mouse IgG(H+L), F(ab’)\u003csub\u003e2\u003c/sub\u003e fragment (Alexa Fluor\u003csup\u003e®\u003c/sup\u003e 488 Conjugate)\u0026nbsp;(catalog no.\u0026nbsp;4408S),\u0026nbsp;Anti-rabbit IgG (H+L), F(ab') 2 Fragment (Alexa Fluor\u003csup\u003e®\u003c/sup\u003e 594 Conjugate) (catalog no.\u0026nbsp;8889S) and DAPI (catalog no.\u0026nbsp;4083S) were purchased from CST.\u0026nbsp;Anti-β-Actin mouse monoclonal antibody (1C7) was purchased from Abbkine (catalog\u0026nbsp;no. A01010).\u0026nbsp;HRP-conjugated goat anti-rabbit IgG (catalog no.\u0026nbsp;BF03008) and goat anti-mouse IgG (catalog no.\u0026nbsp;BF03001) were purchased from BIODRAGON.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConstruction of MGF-deleted ASFV mutants\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic diagram depicting the deletion of MGF100 and MGF300 families was shown in Figure 3A and Figure 4A, respectively. In the case of MGF100 family, as MGF100-1R and MGF100-2L/3L are distributed at different genomic regions,\u0026nbsp;ASFV-Δ100\u0026nbsp;is constructed by replacing MGF100-1R with the reporter gene EGFP under the control of ASFV VP72 promoter as well as replacing MGF100-2L/3L with VP72-mCherry reporter cassette. For MGF300 family, as all the members cluster at the same region,\u0026nbsp;ASFV-Δ300\u0026nbsp;is constructed by the deletion of MGF300 family and insertion of the VP72-mCherry reporter cassette.\u0026nbsp;The resulting virus from each homologous recombination event was purified by\u0026nbsp;first isolating sing cells expressing the expected fluorescent proteins via fluorescence-activated cell sorting (FACS) and later seeding them into fresh BMDMs. All the recombinant viruses undergo at least 10 rounds of single cell sorting combined with limiting dilutions.\u0026nbsp;The virus obtained from the last round of purification\u0026nbsp;procedure\u0026nbsp;was\u0026nbsp;proliferated\u0026nbsp;in BMDMs to\u0026nbsp;generate virus stocks. To ensure the absence of parental\u0026nbsp;ASFV\u0026nbsp;CN/GS/2018 and the desired\u0026nbsp;complete\u0026nbsp;deletions\u0026nbsp;of targeted genes\u0026nbsp;in each recombinant\u0026nbsp;mutant\u0026nbsp;genome, virus DNA was extracted\u0026nbsp;and subject to\u0026nbsp;PCR\u0026nbsp;as well as\u0026nbsp;whole genome\u0026nbsp;sequencing\u0026nbsp;for further purity confirmation. The protocol was described previously with minor modifications\u0026nbsp;\u003csup\u003e5, 20-22\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNext-generation sequencing of ASFV genomes.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMDMs were seeded in 96-well plates and infected with ASFV-Δ100 and ASFV-Δ300 respectively (MOI = 1). At 72 hpi ASFV genome was extracted from the cell cultures following the protocol by E.Z.N.A.\u003csup\u003e®\u003c/sup\u003e Tissue DNA Kit (OMEGA, catalog no. D3396-02). Full-length whole genome sequencing was performed using the MGI SEQ 2000 platform and annotated based on NCBI NT (de redundant nucleotide Library) and NR (de redundant amino acid Library) databases. Sequencing depth and genome coverage were systematically analyzed using the MGI SEQ 2000 platform.\u0026nbsp;The results were defined as reliable when target regions achieved a mean depth of 30,000X, as shown in Figure\u0026nbsp;S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVirus titration\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild-type ASFV CN/GS/2018 and each mutant virus was quantified by using the hemadsorption (HAD) assay as described previously with minor modifications \u003csup\u003e23\u003c/sup\u003e. BMDMs were seeded in 96-well plates. The virus suspensions were 10 times serially diluted and seeded to the plates. The HAD results were recorded on day 5 post-inoculation (p.i.), and 50% HAD doses (HAD\u003csub\u003e50\u003c/sub\u003e) were calculated by using the method of Reed and Muench \u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGrowth kinetics of ASFV in\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eBMD\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eMs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMDMs were grown in 96-well cell culture plates and infected with either parental strains (ASFV CN/GS/2018) or gene-deleted mutant strains (ASFV-Δ100 and ASFV-Δ300) at an MOI of 0.1. The samples were harvested at 2, 12, 24, 36, 48, 72 and 96 hours postinfection (hpi) and titrated by HAD assay.\u0026nbsp;The results were depicted as multi-step growth curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBiosafety statement and facility\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments with live ASFVs were carried out within the biosafety level 3 (BSL-3) facilities in Lanzhou Veterinary Research Institute (LVRI), CAAS approved by the Ministry of Agriculture and Rural Affairs and China National Accreditation Service for Conformity Assessment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimal study\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFourteen ASFV-free Large White outbred pigs typically weighing about 30 kg were obtained from a licensed livestock farm and were randomly divided into two groups housed in two separated rooms in Biosafety Level 3 laboratory (BSL-3) in Lanzhou Veterinary Research Institute (LVRI), CAAS. One group (ear tag No. 5400, 5401, 5410, 5415, 5418, 5422 and 5437) were inoculated intramuscularly (IM) with ASFV-Δ100 (10\u003csup\u003e6.0\u003c/sup\u003e HAD\u003csub\u003e50\u003c/sub\u003e/pig) referring to as ASFV-Δ100 group, whereas the other group (ear tag No. 5412, 5414, 5426, 5427, 5432, 5433 and 5386) were administered with the same dose of ASFV-Δ300 referring to as ASFV-Δ300 group. Following 21 days vaccination, 2 out of 7 pigs died in ASFV-Δ300 group while all the 7 pigs survived in ASFV-Δ100 group. In the lethal challenge experiment, all 5 residual pigs in ASFV-Δ300 group were challenged IM with a lethal dose (10\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e.0\u003c/sup\u003e HAD\u003csub\u003e50\u003c/sub\u003e)\u0026nbsp;of\u0026nbsp;highly virulent\u0026nbsp;ASFV\u0026nbsp;CN/GS/2018 strain. In order to compare the protective rate in two groups, 5 out of 7 pigs from\u0026nbsp;ASFV-Δ100 group\u0026nbsp;were randomly selected for lethal challenge experiment.\u0026nbsp;Daily monitoring was conducted for each pig, which included recording rectal temperatures and observing the clinical signs of each pig, such as lethargy, anorexia, depression, vomiting, fever, skin hemorrhages, bloody diarrhea, and joint swelling. In addition, serum was collected every 2 days\u0026nbsp;\u003csup\u003e25\u003c/sup\u003e, as were oral swabs, nasal swabs, and fecal swab samples. From each necropsied pig, tissues and organs (heart, liver, spleen, lung, kidney, submandibular lymph nodes, hepatogastric lymph nodes and mesenteric lymph nodes) were collected for viral load detection using qPCR and\u0026nbsp;immuno-histochemistry\u0026nbsp;examination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnesthesia Procedure\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the scientific or moderate severity humane endpoint, as defined in the project license, or at the end of animal experimentation, pigs were properly restrained using a snatch. Intramuscular administration of an overdose of Zoletil\u003csup\u003e®\u003c/sup\u003e50 (Virbac, catalog no. 50) was performed at 16 mg per kilogram of body weight. Monitoring of anesthesia depth is performed every 5-10 min. Once the animals demonstrated muscle relaxation, absence of movements, absence of palpebral and loss of consciousness \u003csup\u003e26\u003c/sup\u003e, they are subjected to euthanasia. All anesthesia and euthanasia procedures in this study were in compliance with animal welfare requirements and were strictly conducted in accordance with the \"Standard Operating Procedures for Anesthesia and Euthanasia of Laboratory Animals\" (Document No.: LVRI/HL/SY024-03-03), issued by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLaser confocal microscopy.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the analysis of subcellular localization of MGF100 and MGF300 proteins, HEK293T cells seeded on\u0026nbsp;glass bottom cell culture dishes (NEST, catalog no. 801001)\u0026nbsp;were transfected with overexpression plasmids or backbone vector for 30 h. Then, the cells were incubated with the relevant antibodies and examined with a Zeiss LSM 880 laser-scanning confocal microscope as described previously \u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blotting\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCultured cells were lysed in loading buffer containing 1 × SDS, heated at 100\u0026nbsp;℃\u0026nbsp;for 10 min, then loaded onto SDS-PAGE gel. Then proteins were further electrophoretically transferred onto the polyvinylidene difluoride (PVDF) membrane (Merck-Millipore, catalog no. ISEQ00010) for 2 h with an electric current of 250 mA. Subsequently, the membrane was blocked with a mixture of 5 ml TBS blocking buffer (5% skimmed milk) for 1 h, followed by overnight incubation with primary antibodies at 4\u0026nbsp;℃. The membrane was washed three times and then incubated with appropriate secondary antibody for 1 h. After washing three times, proteins were detected with Enhanced Chemiluminescent (NCE biotech, catalog no. P10300) \u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTransmission electron microscopy\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor transmission electron microscopy (TEM) analysis of virus morphogenesis, BMDM cells were infected with different mutant strains for 30 h and fixed with 2.5% glutaraldehyde (Solarbio, catalog no. P1126) in 0.1 M phosphate buffer for 1 h at RT. Cells were then post-fixed with 2% osmium tetroxide and embedded in epoxy according to standard procedures. After polymerization, about 80 nm-thick sections were obtained and stained with uranyl acetate and lead citrate as previously described \u003csup\u003e29\u003c/sup\u003e. Samples were observed under the JEM-1400 transmission electron microscope operated at 80 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eqPCR assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eASFV DNA was extracted from BMDMs or organ tissues by using E.Z.N.A.\u003csup\u003e®\u003c/sup\u003e Tissue DNA Kit (OMEGA, catalog no.\u0026nbsp;D3396-02) according to the manufacture’s protocols. The qPCR was carried out on a QuantStudio system (Applied Biosystems, USA) by using\u0026nbsp;Pro taq HS premix probe qPCR kit (ACCURATE BIOLOGY AG, catalog no. AG11704).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRNA-seq analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMDM cells were mock infected or infected with WT ASFV, ASFV-Δ100 and ASFV-Δ300 mutants for indicated time periods (18 and 30 hpi). Cells were harvested for RNA extraction using SteadyPure Universal RNA Extraction Kit (ACCURATE BIOLOGY AG, catalog no. AG21017). The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The original results were analyzed on the cloud platform of Oebiotech (https://cloud.oebiotech.com/#/home). The P values were adjusted using the Benjamini-Hochberg method. A corrected P value of\u0026nbsp;\u0026lt;\u0026nbsp;0.05 and absolute fold change (|log2FC|)\u0026nbsp;of \u0026gt;2 were set as the thresholds for significantly differential gene expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistic\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eal\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eanalysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as Mean ± SEM. Comparisons between groups were performed with Mann-Whitney test using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered significant at a \u003cem\u003ep\u003c/em\u003e value less than 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCompliance and ethics\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003eAll animal experiments related to ASFV were conducted in compliance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology (China). The protocols were approved by the Committee on Animal Research and Ethics of Lanzhou Veterinary Research Institute (LVRI), Chinese Academy of Agricultural Sciences (CAAS) and Ethics Committee for Animal Experimentation of Gansu Province, China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThis work was supported by the open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2024KJ14), the National Key R\u0026amp;D Program of China (2021YFD1800100), the Fundamental Research Funds for the Central Universities, China Agriculture Research System of Ministry of Finance and Ministry of Agriculture and Rural Affairs (CARS-35), Project of National Center of Technology Innovation for Pigs (NCTIP-XD/C03), Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSLPDCP-202302 and CAAS-ASTIP-2024-LVRI), Science and Technology Program of Gansu Province (24JRRA01), the China Postdoctoral Science Foundation (2023M733817) and the Postdoctoral Fellowship Program (Grade B) of China Postdoctoral Science Foundation (GZB20230857).\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eW.D., F.X. and H.Z. put forward the concept; F.X. and W.D. wrote the original manuscript text; W.D. reviewed and edited; F.X., T.L. and Y.D. prepared figures 1-3; F.X., M.D., H.L., Z.S., H.T. and J.H. prepared figures 4-7; F.X. and W.D. prepared figure 8; H.Z., M.D. and F.X. acquired fundings. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSpinard, E.et al. Full genome sequence for the African swine fever virus outbreak in the Dominican Republic in 1980. \u003cem\u003eScientific reports\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 1024 (2023).\u003c/li\u003e\n\u003cli\u003eWang, N.et al. Architecture of African swine fever virus and implications for viral assembly. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e366\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 640-644 (2019).\u003c/li\u003e\n\u003cli\u003eTao, D.et al. One year of African swine fever outbreak in China. \u003cem\u003eActa Trop\u003c/em\u003e \u003cstrong\u003e211\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 105602 (2020).\u003c/li\u003e\n\u003cli\u003eZhu, Z.et al. Classification and characterization of multigene family proteins of African swine fever viruses. \u003cem\u003eBrief Bioinform\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2021).\u003c/li\u003e\n\u003cli\u003eO'donnell, V.et al. African Swine Fever Virus Georgia Isolate Harboring Deletions of MGF360 and MGF505 Genes Is Attenuated in Swine and Confers Protection against Challenge with Virulent Parental Virus. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e89\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 6048-56 (2015).\u003c/li\u003e\n\u003cli\u003eReis, A. L.et al. Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 4698-4705 (2016).\u003c/li\u003e\n\u003cli\u003eFan, W.et al. Synergistic effect of the responses of different tissues against African swine fever virus. \u003cem\u003eTransbound Emerg Dis\u003c/em\u003e \u003cstrong\u003e69\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e e204-e215 (2022).\u003c/li\u003e\n\u003cli\u003eSehl-Ewert, J.et al. Pathology of African Swine Fever in Reproductive Organs of Mature Breeding Boars. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2023).\u003c/li\u003e\n\u003cli\u003eNjau, E. P.et al. The first genotype II African swine fever virus isolated in Africa provides insight into the current Eurasian pandemic. \u003cem\u003eScientific reports\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 13081 (2021).\u003c/li\u003e\n\u003cli\u003eWen, X.et al. Genome sequences derived from pig and dried blood pig feed samples provide important insights into the transmission of African swine fever virus in China in 2018. \u003cem\u003eEmerg Microbes Infect\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 303-306 (2019).\u003c/li\u003e\n\u003cli\u003eZhao, D.et al. Replication and virulence in pigs of the first African swine fever virus isolated in China. \u003cem\u003eEmerg Microbes Infect\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 438-447 (2019).\u003c/li\u003e\n\u003cli\u003eIto, S., Bosch, J., Mart\u0026iacute;nez-Avil\u0026eacute;s, M., and S\u0026aacute;nchez-Vizca\u0026iacute;no, J. M. The Evolution of African Swine Fever in China: A Global Threat? \u003cem\u003eFront Vet Sci\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 828498 (2022).\u003c/li\u003e\n\u003cli\u003eRuiz-Saenz, J.et al. African swine fever virus: A re-emerging threat to the swine industry and food security in the Americas. \u003cem\u003eFrontiers in microbiology\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 1011891 (2022).\u003c/li\u003e\n\u003cli\u003eBorca, M. V.et al. Development of a Highly Effective African Swine Fever Virus Vaccine by Deletion of the I177L Gene Results in Sterile Immunity against the Current Epidemic Eurasia Strain. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2020).\u003c/li\u003e\n\u003cli\u003eWu, P. X.et al. African swine fever virus I177L induces host inflammatory responses by facilitating the TRAF6-TAK1 axis and NLRP3 inflammasome assembly. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e e0208024 (2025).\u003c/li\u003e\n\u003cli\u003eBorca, M. V.et al. ASFV-G-∆I177L as an Effective Oral Nasal Vaccine against the Eurasia Strain of Africa Swine Fever. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2021).\u003c/li\u003e\n\u003cli\u003eVan Den Born, E.et al. African swine fever virus vaccine strain Asfv-G-∆I177l reverts to virulence and negatively affects reproductive performance. \u003cem\u003eNpj Vaccines\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 46 (2025).\u003c/li\u003e\n\u003cli\u003eDiep, N. V.et al. Genotype II Live-Attenuated ASFV Vaccine Strains Unable to Completely Protect Pigs against the Emerging Recombinant ASFV Genotype I/II Strain in Vietnam. \u003cem\u003eVaccines (Basel)\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2024).\u003c/li\u003e\n\u003cli\u003eGao, J. Y.et al. A new and efficient culture method for porcine bone marrow-derived M1-and M2-polarized macrophages. \u003cem\u003eVet Immunol Immunop\u003c/em\u003e \u003cstrong\u003e200\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 7-15 (2018).\u003c/li\u003e\n\u003cli\u003eRathakrishnan, A.et al. Deletion of the K145R and DP148R Genes from the Virulent ASFV Georgia 2007/1 Isolate Delays the Onset, but Does Not Reduce Severity, of Clinical Signs in Infected Pigs. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2021).\u003c/li\u003e\n\u003cli\u003eO'donnell, V.et al. African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e89\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 8556-66 (2015).\u003c/li\u003e\n\u003cli\u003eMonteagudo, P. L.et al. BA71\u0026Delta;CD2: a New Recombinant Live Attenuated African Swine Fever Virus with Cross-Protective Capabilities. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e91\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2017).\u003c/li\u003e\n\u003cli\u003eMalmquist, W. A. and Hay, D. Hemadsorption and cytopathic effect produced by African Swine Fever virus in swine bone marrow and buffy coat cultures. \u003cem\u003eAmerican journal of veterinary research\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 104-8 (1960).\u003c/li\u003e\n\u003cli\u003eN\u0026eacute;meth, B.et al. A systematic review of health economic models and utility estimation methods in schizophrenia. \u003cem\u003eExpert review of pharmacoeconomics \u0026amp; outcomes research\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 267-275 (2018).\u003c/li\u003e\n\u003cli\u003eWeesendorp, E., Stegeman, A., and Loeffen, W. Dynamics of virus excretion via different routes in pigs experimentally infected with classical swine fever virus strains of high, moderate or low virulence. \u003cem\u003eVet Microbiol\u003c/em\u003e \u003cstrong\u003e133\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 9-22 (2009).\u003c/li\u003e\n\u003cli\u003eCostea, R., Ene, I., and Pavel, R. Pig Sedation and Anesthesia for Medical Research. \u003cem\u003eAnimals-Basel\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2023).\u003c/li\u003e\n\u003cli\u003eLi, L. F.et al. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity. \u003cem\u003eJournal of Virology\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 4412-4426 (2016).\u003c/li\u003e\n\u003cli\u003eXu, F.et al. IFIT3 mediated the type I interferon antiviral response by targeting Senecavirus A entry, assembly and release pathways. \u003cem\u003eVet Microbiol\u003c/em\u003e \u003cstrong\u003e275\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 109594 (2022).\u003c/li\u003e\n\u003cli\u003eMatamoros, T.et al. African Swine Fever Virus Protein pE199L Mediates Virus Entry by Enabling Membrane Fusion and Core Penetration. \u003cem\u003eMbio\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e (2020).\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, Multigene family 100, Multigene family 300, Morphogenesis, Innate immune, Protective efficacy","lastPublishedDoi":"10.21203/rs.3.rs-6859140/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6859140/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"African swine fever is a highly contagious and lethal disease of swine, but there are few therapeutic options available for treatment. Therefore, it is urgent to develop safe and effective vaccines. In this regard, we described the differential effect of deletion of whole MGF100 and MGF300 families from Genotype Ⅱ highly virulent strain on virus replication, virulence and induction of protection. The resulting ASFV-Δ100 and ASFV-Δ300 mutants demonstrated reduced growth kinetics in vitro, with the former displaying aberrant virus morphogenesis. The ASFV-Δ100 was efficiently attenuated, whereas the ASFV-Δ300 still retained virulence. In homologous lethal challenge, two mutants achieved the same protection rate, with the former providing more pathological protection on organs. Mechanically, we found that ASFV-Δ100 was capable of inducing more robust innate immune in vitro and more consistent P30 antibody response in vivo. To conclude, the MGF100 family is a potential multigene-deleted target for ASFV live attenuated vaccine development.","manuscriptTitle":"MGF100 but not MGF300 family is a potential multigene-deleted target for ASFV attenuation and live attenuated vaccine development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 19:31:16","doi":"10.21203/rs.3.rs-6859140/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":"e046e92f-93d7-460f-bc74-58f605196cc9","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50574316,"name":"Biological sciences/Microbiology/Vaccines/Live attenuated vaccines"},{"id":50574317,"name":"Biological sciences/Immunology/Infectious diseases/Viral infection"}],"tags":[],"updatedAt":"2025-10-09T07:23:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-01 19:31:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6859140","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6859140","identity":"rs-6859140","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}