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ASFV CN/GS/2018 open reading frame (ORF) MGF-110-9L (abbreviated to 9L) encodes for a 290 amino acids protein and is positioned on the reverse strand between nucleotide position nt11627 and nt12499 of the ASFV CN/GS/2018 genome. To assess the degree of conservation of 9L, amino acid sequences from 10 African, European, and Caribbean pathogenic virus isolates were analyzed. The 290 amino acid 9L protein was detected in most of the isolates. A few iso-lates featured a truncated C-terminus of the sequence (Fig. 1). The results indicated that 9L is highly conserved among the isolates.
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To determine the time course of the transcription of the 9L gene, the expression of 9L at the mRNA level was deter-mined at 3, 6, 9, 12, 15, 18, 21, and 24 h post-infection (h.p.i.). The expression of ASFV protein p30 and p72, which occurs early and late in the virus replication cycle respectively, was determined as a control. The p30 and p72 proteins were expressed at approximately 3 or 10 h.p.i. respectively. The expression pattern of 9L was similar to p30 protein (Fig. 2). These results suggested that the 9L gene is transcribed at an early stage in the virus replication cycle.
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To further determine the role of 9L, an ASFV-Δ9L mutant was generated by genetic modification of the highly viru-lent ASFV CN/GS/2018 isolate. The 9L gene was deleted from the ASFV CN/GS/2018 virus using the CRISPR-Cas9 protocol. The 9L gene was replaced with a cassette con-taining the eGFP fluorescent gene under the control of the ASFV p72 promoter (Fig. 3A). The mutant virus was obtained after 11 rounds of purification. The mutant viruses obtained from the last round of purification were replicated in primary swine macrophage cells to obtain a virus stock. The absence of the 9L gene in the ASFV-Δ9L mutant was confirmed by sequence analyses of both the parental and mutant viruses. Green fluorescence was observed in ASFV-Δ9L-infected PAM cells at 12 h.p.i. (Fig. 3B).
Figure 3. Construction of ASFV-Δ9L virus. A Diagram indicating the position of the 9L open reading frame in the ASFV CN/GS/2018 genome. The donor plasmid with the homologous arms to ASFV CN/GS/2018 and GFP under control of the p72 promoter in the orientation as indicated. The final genomic changes introduced to develop ASFV–Δ9L where the sequence of the donor plasmid GFP reporter was introduced to replace the ORF of 9L as indicated. B Primary PAMs infected with ASFV–Δ9L that expressed green fluorescence are shown.
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To evaluate the growth characteristics of ASFV-Δ9L in vitro, replication of both parental ASFV CN/GS/2018 and ASFV-Δ9L in primary swine macrophages was examined at 0, 12, 24, 36, and 48 h.p.i. (Fig. 4). ASFV-Δ9L virus dis-played significantly slower growth kinetics than that of the parental ASFV CN/GS/2018 virus. ASFV-Δ9L yields were fivefold to tenfold lower than those of the parental virus. These results suggested that deletion of the 9L gene signif-icantly hindered the ability of virus replication in vitro in primary swine macrophage cell cultures.
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To examine the pathogenesis and virulence of ASFV-Δ9L, pigs were inoculated intramuscularly with 10 HAD50 of the parental virus ASFV CN/GS/2018 or ASFV-Δ9L. All five animals inoculated with ASFV CN/GS/2018 displayed increased body temperature at the time of death and dis-played clinical signs associated with the disease, including anorexia, depression, purple skin discoloration, staggering gait, and diarrhea (Table 1). Signs of the disease became progressively aggravated over time and the animals died by 7 or 15 days p.i. Two of five animals inoculated intra-muscularly with ASFV-Δ9L displayed these same symp-toms. However, the other three animals developed fever for only a short time and then the temperature returned to normal (Fig. 5A). In addition, all the sentinel animals remained clinically normal (Fig. 5A).
Virus and dose (HAD50) No. of survivors/total Mean time to death (days ± SD) Fever No. of days to onset (days ± SD) Duration No. of days to onset (days ± SD) Maximum daily temp (℃ ± SD) ASFV CN/GS/2018 0/5 12 (3.34) 9 (3) 3 (0.84) 40.71 (0.66) ASFV-Δ9L 3/5 11 (0) 9 (0) 2 (0) 40.65 (0.81) Table 1. Swine survival and fever response following infection with 10 HAD50 doses of ASFV-Δ9L or parental ASFV CN/GS/2018
Figure 5. Kinetics of body temperature values and virus loads in pigs intramuscularly inoculated with 10 HAD50 of ASFV-Δ9L or 10 HAD50 of ASFV CN/GS/2018. A Kinetics of body temperature values in pigs inoculated intramuscularly with 10 HAD50 of ASFV-Δ9L, mock inoculated (sentinels, shown in red) or 10 HAD50 of parental ASFV. B–C Viral DNA detection in blood (B) and different tissues (C) of pigs. D Histomorphologic change of lung, spleen, kidney, and liver in control pigs, ASFV-infected pigs, or ASFV-Δ9L-infected pigs. E Viral DNA detection of oral and nasal swab. Each curve represents values from individual animals in each group.
Animals infected with ASFV CN/GS/2018 presented with high viral load in blood at the time of death (Fig. 5B). Two of five animals inoculated with ASFV-Δ9L developed pronounced viremia. Their viral load had decreased at the time of death (Fig. 5B). Three of the five animals inocu-lated with ASFV-Δ9L showed remarkably lower virus loads in blood than that of the parental virus (Fig. 5B). The results indicated that the deletion of the 9L gene partly attenuated the virulence of the ASFV CN/GS/2018 strain.
Animals inoculated with ASFV CN/GS/2018 displayed high viral loads in tissue samples for the spleen, lung, kidney, and lymph at the time of death (Fig. 5C). Two of five animals infected with ASFV-Δ9L developed remark-ably high viremia in spleen and lung samples, but not in kidney and mesenteric lymph samples at the time of death (Fig. 5C). Three of five animals inoculated with ASFV-Δ9L displayed remarkably low virus loads in different tissues (spleen, lung, kidney, and lymph) (Fig. 5C). In addition, spleens of pigs that survived ASFV-Δ9L infection were normal compared with those of the control, whereas the spleen of dead pigs after ASFV infection was longer than control spleens (Fig. 5D). Consistently, the lungs of pigs that survived ASFV-Δ9L infection were normal compared with those of the control, whereas the infected pigs that died showed pulmonary congestion compared with control pigs (Fig. 5D). The liver, kidney, and heart were normal in ASFV or ASFV-Δ9L-infected pigs compared with control pigs (Fig. 5D). To investigate viral shedding in the ASFV-Δ9L group, viral loads in nasal and oral swabs were determined. The results showed consistent patterns to those observed in the blood experiment (Fig. 5E).
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Previous studies have shown that there is no definite association between host immune mechanisms mediating protection and virulent strains of ASFV in animals infected with attenuated strains of virus (Ruiz Gonzalvo et al. 1986; Onisk et al. 1994; Oura et al. 2005). In order to detect if ASFV-Δ9L-infected animals induced ASFV-specific anti-body response, we found that the surviving pigs infected with ASFV-Δ9L displayed a gradual increase in p30 anti-body at the late stage of infection and ASFV-infected pigs did not induce p30 antibody response (Fig. 6A). In addi-tion, to explore ASFV-Δ9L-induced immune response in vivo, we detected the lgG and lgM expression in pig serum. The results showed that lgG and lgM antibodies can be detected in the surviving pigs infected with ASFV-Δ9L by 5 to 11 days post-inoculation (Fig. 6B and 6C). The results indicated that the surviving pig infected with ASFV-Δ9L is likely to be associated with increasing host antibody response and immune response.
Figure 6. ASFV p30, lgG, and lgM antibodies were detected by ELISA. A Anti-p30 antibody detected by ELISA in pigs intramuscularly inoculated with 10 HAD50 of ASFV-Δ9L or 10 HAD50 of ASFV CN/GS/2018. B–C lgG and lgM antibodies were detected by ELISA in pigs intramuscularly inoculated with 10 HAD50 of ASFV-Δ9L. Each curve represents values from individual animals in each group.