Porcine primary alveolar macrophages (PAMs) were prepared as described previously (Zhang Y. et al., 2021). The viral titer assay was performed according to the method described by Reed and Muench. The strain ASFV SY18 (GenBank number: MH766894) used is a porcine-derived isolate from the Changchun Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences (Zhou et al., 2018).

Twelve ASFV strains of seven genotypes were selected to perform a multiple sequence alignment of L7L, L8L, L9R, L10L, and L11L fragments. For this purpose, the MAFFT website was used, and the Jalview was used to visualize alignment results.

The recombinant transfer vector (p72EGFPΔL7L) contained a fragment of about 1,200 bp of the gene flanking the L7L gene, and the p72 promoter EGFP gene cassette. p72EGFPΔL8L, p72EGFPΔL9R, p72EGFPΔL10L, and p72EGFPΔL11L were obtained identically. PAM cells (2 × 10⁶) PAM cells were spread into a 6-well plate, the recombinant transfer vector was added to the cells using Jet-Macrophage (Polyplus) transfection reagent, 1 MOI of ASFV SY18 was added after 4 h, and fluorescence was observed after 24 h. The fluorescence intensity was screened, and the purified virus was obtained using the limited dilution method. Polymerase chain reaction was used to identify the ASFV mutants, and specific sequences are shown in Table 1. Recombinant viral DNA was extracted and sent to Novogene (Tianjin, China) for next-generation sequencing.

A total of 2 × 106 PAM cells were spread into 6-well plates, and infected with ASFV SY18, SY18ΔL7L, SY18ΔL8L, SY18ΔL9R, SY18ΔL10L, SY18ΔL11L at an amount of 0.1 MOI. The samples were collected at 2, 12, 24, 48, 72, and 96 h after infection. The samples were then freeze-thawed three times, and the TCID50 of each sample was determined.

Experiments were conducted according to standard procedures approved by the Animal Welfare and Ethics Committee of the Changchun Veterinary Research Institute and the Animal Biosafety Level 3 (ABSL-3) Laboratory.

Indirect ELISA was used to detect anti-p54 antibodies. Anti-p54 antibodies were detected in each serum sample as previously described, and sample optical density (OD) values were measured at 450 nm. Samples were considered antibody positive when the sample OD450 nm/positive control OD450 nm (S/P) was greater than 0.25.

A total of 309 bp in L7L encodes 102-amino-acids, 312 bp in L8L encodes 103, 291 bp in L9R encodes 96, 531 bp in L10L encodes 170, and 282 bp in L11L encodes 93 (Figure 1). They were located between nucleotide positions 180,724 and 181,032 of the reverse strand, 181,246 and 181,557 of the antisense strand, 181,752 and 182,042 of the plus strand, 182,118 and 182,630 of the antisense strand, and 182,869 and 183,150 of the antisense strand of the ASFV SY18 genome (Figure 2). Degree of conservation of L7L, L8L, L9R, L10L, and L11L in ASFV isolates of multiple genotypes. A comparison of the amino acid sequences of the isolate ASFV SY18 with those of genotype II strains isolated from Heilongjiang, China (Pig/HLJ/2018), Georgia (Georgia/2007/1), and Estonia (Estonia/2014) revealed that the amino acid sequences of L7L-L11L were completely identical. The amino acid sequence of L8L was identical in genotype I and II isolates. The amino acid sequences of L7L, L8L, L9R, L10L, and L11L were identical in the same genotype, and a large gap was observed between the different genotypes. The L7L sequence in the Pretoriuskop/96/4 strain (genotype X) was significantly different than those in other strains. The ASFV genotype II is prevalent globally. Positions 1, 17, 21, 24, and 67 of L11L in genotype II ASFV SY18, Pig/HLJ/2018, Georgia/2007/1, and Estonia/2014 included a methionine. However, L11L of OURT88/3, NHV, Benin97/1 (genotype I) had 77 amino acids, L11L of Malawi Lil/20/1 (genotype VIII) has 78, L11L of R8 (genotype IX) had 78, L11L of Pretoriuskop/96/4 (genotype XX) had 78 amino acids. These strains did not include the first 16 amino acids found in L11L of ASFV SY18, with the 17th amino acid in the L11L of ASFV SY18 being the most common initial amino acid (the second occurrence of methionine in the L11L of genotype II). The L11L of Tengani/62 (genotype V) contained 93 amino acids. The L11L of warm baths (genotype III) had the same sequence of amino acids between positions 3 and16 compared to L11L of ASFV SY18, except that the first amino acid was not methionine. Therefore, the first 16 amino acids of L11L in warm baths were also non-functional.

In order to identify the specific virulence genes among L7L-L11L, we designed and constructed single-gene deletion strains (SY18ΔL7L, SY18ΔL8L, SY18ΔL9R, SY18ΔL10L, and SY18ΔL11L). The construction method is shown in Figure 2. Target genes were replaced with a green fluorescent gene cassette containing the p72 promoter using a recombinant method. The recombinant viruses were purified using fluorescence activity. The PCR assay determined that the target bands of the gene were not detected, whereas the ASFV SY18 control showed the target bands, confirming that single-gene deletion viruses missing each target gene were successfully obtained (Figure 3). Comparison of the whole-genome sequences of the ASFV mutant and parental ASFV SY18 using second-generation sequencing revealed that, apart from design changes, no unwanted mutations were detected in the genomes of the constructed mutants.

Macrophages were infected with each deletion strain at an MOI of 0.1, and the parental ASFV SY18 was used as a control. Samples were collected at 2, 12, 24, 48, 72, and 96 h post-infection (hpi). The results shown in Figure 4 revealed no significant differences between the growth curves of single-gene deletion viruses in target cells than those of the parental strains in vitro. There was also no difference in the replication pattern between the single-gene deletion strains, all of which reached the highest titer at 72 hpi. These findings demonstrate that the deletion of L7L, L8L, L9R, L10L, and L11L did not affect the proliferative ability of ASFV in macrophages in vitro.

To assess the virulence of SY18ΔL7L, SY18ΔL8L, SY18ΔL9R, SY18ΔL10L, SY18ΔL11L, and ASFV SY18 (positive control group) for pigs, each group was injected intramuscularly with 10^3.0 TCID₅₀ of virus. The survival and body temperature results are shown in Figures 5A,B and Table 2. Pigs in the ASFV SY18 group became febrile on day 4 after infection, and showed typical symptoms of ASF, and died on day 8 post infection. Pigs in the SY18ΔL7L group showed persistent fever between days 1 and 5. Three of these pigs died on the 9th, 12th, and 13th dpi, and one pig (S7-1) survived during the observation period. Pigs in the SY18ΔL8L group died on days 7 and 8 post-infection, which was consistent with the timing of death in the control group. However, only one animal in the SY18ΔL8L group showed elevated body temperature, whereas the remaining three animals had normal body temperatures. Pigs in the SY18ΔL9R group died 10 to 16 days and pigs in the SY18ΔL10L group died 10 to 13 days post infection, respectively. Deaths in both groups delayed backwards compared with the control. All pigs in the SY18ΔL11L group survived the 21 days of observation, with three pigs experiencing a transient increase in body temperature and one returning to normal temperature after 6 days of fever.

The viral loads in the blood of the experimental animals are shown in Figure 5C. Viral nucleic acids were detected in the blood of all animals on day 3 after infection, except for one pig in the SY18ΔL7L group. On the 3rd day of infection, all pigs in the ASFV SY18 group showed high viral loads in their blood (10^6.19–10^6.86 copies/mL), and pigs in the SY18ΔL8L and SY18ΔL9R groups similarly showed high viremia (10^6.72–10^7.72 copies/mL and 10^6.16–10^7.13 copies/mL). On the 3rd day of infection, pigs in the SY18ΔL7L, SY18ΔL10L, and SY18ΔL11L groups had lower viral loads in their blood compared to the control group (0–10^5.08 copies/mL, 10^3.58–10^7.12 copies/mL, and 10^3.37–10^5.42 copies/mL, respectively). The highest viral levels in the blood of all animals were reached on the 7th dpi. Subsequently, the viral load in the blood of surviving animals tended to decrease. The presence of virus was not detected in the blood of 1 pig without fever (S7-1) in the SY18ΔL7L group on the 3rd day, and the presence of the virus was detected on the 7th day, after which and the virus level was consistently low (10^3.56–10^4.44 copies/mL).

The pigs used in Experiment 1 were dissected, and organs including the heart, liver, spleen, lungs, kidneys, inguinal lymph nodes, thymus, submandibular lymph nodes, colon, bone marrow, muscles, and joint fluids were collected and tested for the presence of the virus. Figure 5D indicates that most animals had the virus in their organs, with the spleen, liver, and bone marrow showing the highest detection rates. In the SY18ΔL11L group, both the detection rate and viral load in the organs were lower compared to other groups. In the SY18ΔL7L group of non-febrile pigs (S7-1), post-dissection testing of tissues showed no viral nucleic acid except in the muscle tissue (10^2.68 copies/mL).

Specific antibody responses in pigs infected with ASFV mutants. To assess the specific immune response, we determined the levels of anti-p54 antibodies in the serum. As shown in Figure 5E, the sera of each animal were negative for anti-p54 antibodies on day 7 of immunization, and those that survived until day 14 (all pigs in the S9-1, S9-4, S7-1 and SY18ΔL11L groups) were positive for anti-p54 antibodies. Pigs S9-1 and S9-4 produced higher levels of anti-p54 antibodies (S/p values of 1.123 and 1.197, respectively), yet died on days 15th and 16th days. The S11-4 pigs in the SY18ΔL11L group expressed lower anti-p54 antibodies than S9-1, S9-4, but the S11-4 pigs survived the observation period. This finding demonstrates that the expression of ASF-specific antibodies is not a marker for animal survival.

To assess the degree of attenuation of SY18ΔL11L, a low dose (10^3.0TCID₅₀) and a high dose (10^6.0TCID₅₀) of SY18ΔL11L were inoculated intramuscularly. Another group of pigs was injected with an equal amount of saline as control (Figures 6A,B; Table 3). All pigs in the low-dose SY18ΔL11L-infected group survived, with two pigs experiencing a transient increase in body temperature and two pigs having persistent fever for 4–5 days. All animals in the high-dose SY18ΔL11L-infected group showed elevated body temperatures (>40.5°C), with symptoms of ASF in H-2 and H-3, which were euthanized on days 11th and 15th days post-inoculated due to the severity of the disease in both animals. Pigs in the control group showed no adverse reactions. To determine the response of the surviving pigs when challenged with the parental virus, a virus provocation test was performed on all pigs that survived the inoculation observation period (Figures 6C,D; Table 4). Each pig was intramuscularly injected with 10^3.0TCID₅₀ of SY18 cells, and observed for 28 days. During the viral challenge, all pigs in the control group had elevated temperatures, developed an ASF clinical reaction, and were euthanized by the 9th day post-challenge (dpc). Pigs inoculated with SY18ΔL11L all survived during the challenge.

Animals inoculated with SY18ΔL11L strain developed viremia on day 7 after immunization (Figure 7A), with the viral load in blood reaching its maximum on day 7 or 14 post-infection, followed by a slow decrease. After ASFV SY18 challenge, control animals exhibited high viral loads in their blood. SY18ΔL11L-infected animals challenged with the parental virus exhibited a steady decline in blood viral load after an increased on day 7. Then, no virus in blood of all but 1 pig on the 28th dpc was be detected (Figure 7B). The ASFV load in the tissues and organs of the animals was measured at the end of the observation period. The results showed that pigs in the control group had higher viral detection in tissues and higher viral loads (Figure 7C). Pigs that died during the immunization period (H2 and H3) had relatively higher virus detection in tissues compared to other pigs immunized with SY18ΔL11L.

The host antibody responses in animals inoculated with SY18ΔL11L are shown in Figure 7D. Animals inoculated with SY18ΔL11L were weakly positive for anti-p54 antibodies in sera of L3 and H4 on day 7 of immunization, and positive for antibodies on day 14, followed by high levels of antibodies in sera thereafter. H3 pigs in the high-dose SY18ΔL11L group died on day 15 even though they were positive for antibodies in serum on day 14 of immunization. After the takedown challenge, pigs in the control group remained negative, and antibodies in pigs inoculated with SY18ΔL11L remained at high levels.