Recombinant Vaccine Strain ASFV-G-Δ9GL/ΔUK Produced in the IPKM Cell Line Is Genetically Stable and Efficacious in Inducing Protection in Pigs Challenged with the Virulent African Swine Fever Virus Field Isolate Georgia 2010

We have previously reported that the recombinant African Swine Fever (ASF) vaccine candidate ASFV-G-Δ9GL/ΔUK efficiently induces protection in domestic pigs challenged with the virulent strain Georgia 2010 (ASFV-G). As reported, ASFV-G-Δ9GL/ΔUK induces protection, while intramuscularly (IM), administered at doses of 104 HAD50 or higher, prevents ASF clinical disease in animals infected with the homologous ASFV g strain. Like other recombinant vaccine candidates obtained from ASFV field isolates, ASFV-G-Δ9GL/ΔUK stocks need to be produced in primary cultures of swine macrophages, which constitutes an important limitation in the production of large virus stocks at the industrial level. Here, we describe the development of ASFV-G-Δ9GL/ΔUK stocks using IPKM (Immortalized Porcine Kidney Macrophage) cells, which are derived from swine macrophages. We show that ten successive passages of ASFV-G-Δ9GL/ΔUK in IPKM cells induced small changes in the virus genome. The produced virus, ASFV-G-Δ9GL/ΔUKp10, presented a similar level of replication in swine macrophages cultures to that of the original ASFV-G-Δ9GL/ΔUK (ASFV-G-Δ9GL/ΔUKp0). The protective efficacy of ASFV-G-Δ9GL/ΔUKp10 was evaluated in pigs that were IM-inoculated with either 104 or 106 HAD50 of ASFV-G-Δ9GL/ΔUKp10. While animals inoculated with 104 HAD50 present a partial protection against the experimental infection with the virulent parental virus ASFV-G, those inoculated with 106 HAD50 were completely protected. Therefore, as was just recently reported for another ASF vaccine candidate, ASFV-G-ΔI177L, IPKM cells are an effective alternative to produce stocks for vaccine strains which only grow in swine macrophages.


Introduction
African Swine Fever virus (ASFV) produces a lethal disease in domestic swine.African Swine Fever (ASF), which severely affects the production of domestic pigs worldwide, is currently widely distributed, being present in Africa, Asia, Europe, and, recently, in the Caribbean region [1][2][3].ASFV is a large and structurally complex virus.Its genome is composed of double-stranded DNA of approximately 180-190 kilobase pairs which encode more than 160 genes [4][5][6].
Pathogens 2024, 13, 319 2 of 9 Although the development of vaccines to prevent ASF was attempted for years [7], commercial vaccines were not available until very recently.Therefore, the control of the disease was based on the elimination of infected and susceptible animals, as well as the restricted mobility of infected herds [8,9].
Among those recombinant live attenuated viruses is the ASFV-G-∆9GL/∆UK [23].ASFV-G-∆9GL/∆UK was developed by a double gene deletion in the genome of the virulent isolate Georgia 2010 (ASFV-G) of the 9GL and the UK genes.This recombinant virus was shown to have no residual virulence in domestic pigs even when parenterally inoculated at doses as high as 10 6 HAD 50 [23].Importantly, ASFV-G-∆9GL/∆UK efficaciously induces protection in vaccinated pigs against the experimental infection using the highly virulent homologous ASFV g [23].ASFV-G-∆9GL/∆UK was reported to produce protection as early as 2 weeks after a single vaccine dose of at least 10 4 HAD 50 [23].ASFV-G-∆9GL/∆UK was developed, purified, and stock was generated using primary cell cultures of swine macrophages.The use of primary cell cultures is a difficulty in the necessary scaling up of vaccine production with commercial purposes.As an alternative, the adaptation of ASFV to grow in a cell line is usually accompanied by significant and undesirable modifications in the virus genome that appear during the process of the virus adaptation [24].Here, we report the production of ASFV-G-∆9GL/∆UK stocks utilizing the cell line IPKM as growing substrate [25].It is demonstrated that ASFV-G-∆9GL/∆UK replicates efficiently in IPKM cells incorporating minimal genomic changes.In addition, pigs were partially and fully protected against the challenge with the virulent parental virus when they received a single vaccine dose containing 10 6 or 10 4 HAD 50 of ASFV-G-∆9GL/∆UK, respectively.Consequently, IPKM cells can be an option for the massive production of ASFV-G-∆9GL/∆UK stocks.

Viruses and Cells
The ASFV-G-∆9GL/∆UK vaccine was produced in the Plum Island Animal Disease Center, as previously reported [13].Primary cultures of peripheral blood swine macrophages were produced, as reported earlier [22].Macrophages were used at a final concentration of 5 × 10 6 cells/mL to coat 96 or 6 well plates, as needed.Titrations of ASFV-G-∆9GL/∆UK and its parental field isolate ASFV g were implemented in primary swine macrophage cultures as earlier reported [22] and summarized below.The immortalized porcine kidney macrophage derived cell line IPKM [25] was kindly provided by Dr Kokuho Takehiro from the National Institute of Animal Health of Japan.IPKM cells were grown under culture conditions, as previously described [25].ASFV-G-∆9GL/∆UK (ASFV-G-∆9GL/∆UKp0) and the parental ASFV g (ASFV-Gp0) were sequentially passed 10 times in IPKM cultures (MOI = 1), producing ASFV-G-∆9GL/∆UKp10 and ASFV-Gp10, respectively.Every passage proceeded until cytopathic effect reached approximately 80% of the cells.Intermediate virus stocks were prepared by one freezing and thawing step, the clarification of the obtained cell suspension by centrifugation, and their titration on primary swine macrophage cultures, as described below.Growth kinetics studies comparing different ASFV-G-∆9GL/∆UKp0 and p10 stocks and those of ASFV-Gp0 and p10 were performed at a MOI of 0.01 HAD 50 using previously published protocols [22].Virus yields were titrated at different times post-infection on swine macrophages.The presence of virus-infected cells was evaluated by hemadsorption (HA), as previously reported, and determined by the Reed and Muench method [26].

Evaluation of ASFV-G-∆9GL/∆UKp10 Efficacy in Domestic Pigs
The ability of ASFV-G-∆9GL/∆UKp10 to protect domestic pigs against infection with the highly virulent ASFV g was assessed in 35-40 kg crossbreed pigs.Groups of 5 animals were inoculated intramuscularly (IM), receiving either 10 4 or 10 6 HAD 50 of ASFV-G-∆9GL/∆UKp10.Another group of pigs was mock-inoculated with the culture medium.The presence of the ASF clinical disease as well as changes in body temperature reads were recorded daily for 28 days.The harvesting of blood samples (in heparinized blood collection tubes) to quantify the presence of the virus was scheduled to be performed at days 0, 4, 7, 11, 14, 21, and 28 post-infection (pi).By day 28 pi, all groups of pigs were IM-challenged with 10 2 HAD 50 of ASFV-G.Animals were monitored and sampled, as described above, until day 21 post challenge (pc).The experiments with animals were performed under biosafety level 3 conditions in the animal facilities at Plum Island Animal Disease Center, strictly following a protocol approved by the Institutional Animal Care and Use Committee (225.06-19-R_090716,approved on 9 June 2019).

Effect of Sequential Passages of ASFV-G-∆9GL/∆UK in IPKM Cells
In most cases, the growth of an ASFV strain in a cell line requires a process of adaptation, usually accompanied by important genomic changes.To evaluate the capability of IPMK cells to allow the growth of ASFV-G-∆9GL/∆UK without a preliminary period of adaptation, ASFV-G-∆9GL/∆UK was subjected to a set of 10 successive passages in IPMK cells.In these passages, infection was performed using an MOI of 1 (the initial and all intermediate stocks' titers were calculated based on titrations implemented in swine macrophages).
All virus passages in IPKM cells were halted when the cytopathic effect reached approximately 80% of the cells.At that point, cultures were frozen and each of the intermediate stocks prepared, as described in Materials and Methods.A similar experiment was also performed in parallel with the parental virus ASFV-G.
The results showed that the virus yields of the recombinant ASFV-G-∆9GL/∆UK as well as ASFV g remained without large fluctuations during the 10 passages in the IPKM cells (Figure 1).Virus yield along the passages remained within a range, with titer values of 10 5.4-7.3HAD 50 /mL for ASFV-G-∆9GL/∆UK and between 10 5.8 to 10 8 HAD 50 /mL for ASFV-G.Therefore, the two viruses effectively replicate in the IPKM cells without needing a clear phase of adaptation.These data results support results already reported, showing that several ASFV isolates (Armenia07, Kenya05/Tk-1, Espana75 and Lisbon60) easily replicate in IPKM cells [28], reaching virus yields similar to those obtained in the primary cultures of swine macrophages.

Genomic Stability of ASFV-G-∆9GL/∆UK in IPKM Cells
To assess the genomic stability of ASFV-G-∆9GL/∆UK during the 10 successive passages in IPKM cells (ASFV-G-∆9GL/∆UK p10), ASFV-G-∆9GL/∆UKp10 was sequenced and the result was compared to that of the ASFV-G-∆9GL/∆UKp0.Just one mutation of high confidence (over 70% of the reads at that position contained the SNP) was observed, at nucleotide position 127,166 within the CP530R gene, where a G-to-A mutation produced a Gly-to-Ser substitution.This gene encodes for the virus polyprotein pp62, described as being essential to the processes of virus core development [29].It is not clear if the glycine-to-serine amino acid substitution detected in the ASFV-G-∆9GL/∆UKp10 may affect the function of pp62L protein.Similar results in terms of the genome stability of ASFV strains sequentially passed in IPKM cells have been reported for Armenia 2007, ASFV g isolates and the vaccine candidate ASFV-G-∆I177L [27,28].

Genomic Stability of ASFV-G-Δ9GL/ΔUK in IPKM Cells
To assess the genomic stability of ASFV-G-Δ9GL/ΔUK during the 10 successive passages in IPKM cells (ASFV-G-Δ9GL/ΔUK p10), ASFV-G-Δ9GL/ΔUKp10 was sequenced and the result was compared to that of the ASFV-G-Δ9GL/ΔUKp0.Just one mutation of high confidence (over 70% of the reads at that position contained the SNP) was observed, at nucleotide position 127,166 within the CP530R gene, where a G-to-A mutation produced a Gly-to-Ser substitution.This gene encodes for the virus polyprotein pp62, described as being essential to the processes of virus core development [29].It is not clear if the glycine-to-serine amino acid substitution detected in the ASFV-G-Δ9GL/ΔUKp10 may affect the function of pp62L protein.Similar results in terms of the genome stability of ASFV strains sequentially passed in IPKM cells have been reported for Armenia 2007, ASFV g isolates and the vaccine candidate ASFV-G-ΔI177L [27,28].

Evaluation of the Virus Replication of the Recombinant ASFV-G-Δ9GL/UK in IPKM Cells
The growth kinetics of ASFV-G-Δ9GL/ΔUKp10 on IPKM cells was assessed by comparison with that of the original parental virus stock, ASFV-G-Δ9GL/ΔUKp0.In addition, the replication ability of both ASFV-Gp0 and ASFV-Gp10 was assessed.The study was conducted as a multistep growth assay on primary swine macrophage cultures.Infections were performed (MOI = 0.01) and virus yields assessed at 2, 24, 48, 72, and 96 h pi by titration performed in swine macrophages.

Evaluation of the Virus Replication of the Recombinant ASFV-G-∆9GL/UK in IPKM Cells
The growth kinetics of ASFV-G-∆9GL/∆UKp10 on IPKM cells was assessed by comparison with that of the original parental virus stock, ASFV-G-∆9GL/∆UKp0.In addition, the replication ability of both ASFV-Gp0 and ASFV-Gp10 was assessed.The study was conducted as a multistep growth assay on primary swine macrophage cultures.Infections were performed (MOI = 0.01) and virus yields assessed at 2, 24, 48, 72, and 96 h pi by titration performed in swine macrophages.

ASFV-G-∆9GL/∆UKp10 Replication in Experimentally Infected Domestic Pigs
To assess the ability of ASFV-G-∆9GL/∆UKp10 to replicate in domestic pigs and efficaciously protect them against infection with the virulent ASFV g isolate, two groups (n = 5) of 35-40 kg pigs were IM-inoculated with either 10 4 or 10 6 HAD 50 .These doses of the vaccine were chosen since, when originally reported [23], ASFV-G-∆9GL/∆UK demonstrated the ability to protect against ASFV challenge.A group of similar characteristics was used as a control.The potential presence of clinical signs related to ASF was checked daily for 28 days after inoculation.Results demonstrated that all animal groups remained clinically normal for the whole observational period (Figure 3), indicating that ASFV-G-∆9GL/∆UKp10 continues to be fully attenuated when inoculated in domestic pigs, even at doses as high as 10 6 HAD 50 .indicates significant differences between ASFV-G-Δ9GL/ΔUKp0 and ASFV-G-Δ9GL/ΔUKp10 at specific time points.Differences were inferred by the unpaired t test using the two-stage set up (Benjamine, Krieger and Yekutieli) method.The reliability of multiple comparisons was evaluated by the false discovery rate method (FDR), considering a q-value < 0.05.

ASFV-G-Δ9GL/ΔUKp10 Replication in Experimentally Infected Domestic Pigs
To assess the ability of ASFV-G-Δ9GL/ΔUKp10 to replicate in domestic pigs and efficaciously protect them against infection with the virulent ASFV g isolate, two groups (n = 5) of 35-40 kg pigs were IM-inoculated with either 10 4 or 10 6 HAD50.These doses of the vaccine were chosen since, when originally reported [23], ASFV-G-Δ9GL/ΔUK demonstrated the ability to protect against ASFV challenge.A group of similar characteristics was used as a control.The potential presence of clinical signs related to ASF was checked daily for 28 days after inoculation.Results demonstrated that all animal groups remained clinically normal for the whole observational period (Figure 3), indicating that ASFV-G-Δ9GL/ΔUKp10 continues to be fully attenuated when inoculated in domestic pigs, even at doses as high as 10 6 HAD50.In vitro growth kinetics of ASFV-G-∆9GL/∆Ukp10 in primary swine macrophages (MOI = 0.01).Samples were taken at the indicated time points and titrated in swine macrophages.Titrations were performed in swine macrophages.Data represent means and standard deviations from two independent experiments.The sensitivity of virus detection is ≥log10 1.8 HAD 50 /mL.The symbol (*) indicates significant differences between ASFV-G-∆9GL/∆UKp0 and ASFV-G-∆9GL/∆UKp10 at specific time points.Differences were inferred by the unpaired t test using the two-stage set up (Benjamine, Krieger and Yekutieli) method.The reliability of multiple comparisons was evaluated by the false discovery rate method (FDR), considering a q-value < 0.05.

Figure 2.
In vitro growth kinetics of ASFV-G-Δ9GL/ΔUkp10 in primary swine macrophages (MOI = 0.01).Samples were taken at the indicated time points and titrated in swine macrophages.Titrations were performed in swine macrophages.Data represent means and standard deviations from two independent experiments.The sensitivity of virus detection is ≥log10 1.8 HAD50/mL.The symbol (*) indicates significant differences between ASFV-G-Δ9GL/ΔUKp0 and ASFV-G-Δ9GL/ΔUKp10 at specific time points.Differences were inferred by the unpaired t test using the two-stage set up (Benjamine, Krieger and Yekutieli) method.The reliability of multiple comparisons was evaluated by the false discovery rate method (FDR), considering a q-value < 0.05.

ASFV-G-Δ9GL/ΔUKp10 Replication in Experimentally Infected Domestic Pigs
To assess the ability of ASFV-G-Δ9GL/ΔUKp10 to replicate in domestic pigs and efficaciously protect them against infection with the virulent ASFV g isolate, two groups (n = 5) of 35-40 kg pigs were IM-inoculated with either 10 4 or 10 6 HAD50.These doses of the vaccine were chosen since, when originally reported [23], ASFV-G-Δ9GL/ΔUK demonstrated the ability to protect against ASFV challenge.A group of similar characteristics was used as a control.The potential presence of clinical signs related to ASF was checked daily for 28 days after inoculation.Results demonstrated that all animal groups remained clinically normal for the whole observational period (Figure 3), indicating that ASFV-G-Δ9GL/ΔUKp10 continues to be fully attenuated when inoculated in domestic pigs, even at doses as high as 10 6 HAD50.The ability of ASFV-G-∆9GL/∆UKp10 to replicate after its inoculation was assessed by quantifying its viremia titers.The pattern of viremia in the inoculated animals was heterogeneous in both groups (Figure 4).In the group inoculated with 10 4 HAD 50 /mL, two of the animals showed undetectable levels of viremia throughout the observational period of 28 days pi.Another animal remained negative until day 21 pi, when it showed low titers (10 2.3 HAD 50 /mL) that remained at that level until the day of challenge (28 days pi).Viremia in the fourth animal inoculated with 10 4 HAD 50 /mL remained undetectable until day 11 pi, when high titers were detected (10 5.05 HAD 50 /mL), and it showed variable titers (ranging between 10 5.8-3.8HAD 50 /mL) until day 28 pi.The fifth animal in the group showed relatively high viremia titers (ranging between 10 4.3-6.3HAD50/mL) from day 7 pi until the challenge day.day 11 pi, when high titers were detected (10 5.05 HAD50/mL), and it showed variab (ranging between 10 5.8-3.8HAD50/mL) until day 28 pi.The fifth animal in the group s relatively high viremia titers (ranging between 10 4.3-6.3HAD50/mL) from day 7 pi u challenge day.With the exception of one pig, which presented undetectable viremia titers the 28 days following inoculation, all animals receiving 10 6 HAD50/mL of A Δ9GL/ΔUKp10 showed low-to-medium titers by day 7 pi (10 2.05-4.3HAD50/mL).these animals evolved, showing medium-to-high viremia titers (ranging between HAD50/mL) by days 11 to 21 pi, and lower titers by the day of challenge HAD50/mL).The remaining two pigs of the group presented medium titers ( HAD50/mL) on days 11 and 21 pi and undetectable levels by day 28 pi.
These levels of replication are clearly lower than viremia titers detected in p inoculated with similar doses of the original stock ASFV-G-Δ9GL/ΔUK (A Δ9GL/ΔUKp0) [23].

Evaluation of the Protective Effect of ASFV-G-Δ9GL/ΔUKp10 on Experimental Inocula with the Parental ASFV-G
The ability of ASFV-G-Δ9GL/ΔUK to protect animals against the experimental in with the parental virus ASFV g was evaluated by IM challenging the animals pre inoculated with ASFV-G-Δ9GL/ΔUK 28 days later with 10 2 HAD50 of ASFV-G.Th vaccinated control pigs were similarly infected.
The mock-inoculated pigs presented initial signs of ASF by days 3-4 post ch (dpc), quickly getting worse with all of them euthanized due to the severity of the signs by day 4, one of them early, and the remaining four on day 5 pc (Figures 3 Conversely, all the animals inoculated with 10 6 HAD50 of ASFV-G-Δ9GL/ΔUK mained clinically normal during the observation period, except for two, presenti period of transient mild body temperature elevation without any other addition With the exception of one pig, which presented undetectable viremia titers during the 28 days following inoculation, all animals receiving 10 6 HAD 50 /mL of ASFV-G-∆9GL/∆UKp10 showed low-to-medium titers by day 7 pi (10 2.05-4.3HAD50/mL).Two of these animals evolved, showing medium-to-high viremia titers (ranging between 10 4.55-5.8HAD50/mL) by days 11 to 21 pi, and lower titers by the day of challenge (10 2.3-2.8HAD50/mL).The remaining two pigs of the group presented medium titers (10 2.05-4.3HAD50/mL) on days 11 and 21 pi and undetectable levels by day 28 pi.

Evaluation of the Protective Effect of ASFV-G-∆9GL/∆UKp10 on Experimental Inoculation with the Parental ASFV-G
The ability of ASFV-G-∆9GL/∆UK to protect animals against the experimental infection with the parental virus ASFV g was evaluated by IM challenging the animals previously inoculated with ASFV-G-∆9GL/∆UK 28 days later with 10 2 HAD 50 of ASFV-G.The mock vaccinated control pigs were similarly infected.
The mock-inoculated pigs presented initial signs of ASF by days 3-4 post challenge (dpc), quickly getting worse with all of them euthanized due to the severity of the clinical signs by day 4, one of them early, and the remaining four on day 5 pc (Figures 3 and 5).Conversely, all the animals inoculated with 10 6 HAD 50 of ASFV-G-∆9GL/∆UKp10 remained clinically normal during the observation period, except for two, presenting one period of transient mild body temperature elevation without any other additional sign associated with ASF (Figures 3 and 5).Animals in the group receiving 10 4 HAD 50 of ASFV-G-∆9GL/∆UKp10 presented a heterogenous response after the challenge.Two of the animals presented initial signs of disease by days 4-5 post challenge, followed by a quick evolution to the severe clinical form of the disease and the need to be euthanized by days 6-7 pc.The remaining three animals did not show any clinical sign associated with ASF during the 21-day observational period, with the exception of very mild and transitory elevation in body temperature (Figures 3 and 5).
G-Δ9GL/ΔUKp10 presented a heterogenous response after the challenge.Two of the animals presented initial signs of disease by days 4-5 post challenge, followed by a quick evolution to the severe clinical form of the disease and the need to be euthanized by days 6-7 pc.The remaining three animals did not show any clinical sign associated with ASF during the 21-day observational period, with the exception of very mild and transitory elevation in body temperature (Figures 3 and 5).Viremias in the mock-vaccinated animals increased after the challenge.In all but one animal, titers ranged between 10 6.5 and 10 8.3 HAD50/mL by day 4 pc and remained at that level until all the animals were euthanized.The remaining animal in this group presented a low (10 2.5 HAD50) viremia by day 4 pc, increasing to 10 5 HAD50 at the time of the euthanasia (Figure 4).Animals inoculated with 10 6 HAD50 of ASFV-G-Δ9GL/ΔUKp10 presented low viremias after the challenge, ranging between 10 1.8 and 10 3 HAD50/mL.Viremia in most of these animals, except for one, decreased until it hit undetectable levels (≤10 1.8 HAD50/mL) before the end of the 21-day pc observational period.Animals receiving 10 4 HAD50 of ASFV-G-Δ9GL/ΔUKp10 had a viremia pattern in accordance with their clinical status.The three animals surviving the challenge presented low viremias after the challenge, ranging between undetectable (≤10 1.8 HAD50/mL) and 10 2.8 HAD50/mL.The two animals that succumbed to the challenge quickly presented medium-to-high viremias by the day they were euthanized because of the severity of the disease (10 4.5 and 10 7.8 HAD50/mL, respectively).Viremia in two of the remaining three animals decreased until it reached undetectable levels.
It is shown that the recombinant vaccine candidate ASFV-G-Δ9GL/ΔUK may be produced in the cell line IPKM.A stock virus produced after 10 sequential passages in these cells is still safe and efficacious in protecting animals against the infection with the virulent ASFV g when used at a dose of 10 6 HAD50/mL.It is not clear why, though no main genetic changes are observed in the ASFV-G-Δ9GL/ΔUKp10 genome; this strain is slightly less efficacious than the parental ASFV-G-Δ9GL/ΔUK, which is able to protect pigs at doses of 10 4 HAD50/mL.
As it was previously described for the ASFV-G-ΔI177L vaccine strain [27], these results demonstrate the feasibility of employing the IPKM cell line to produce ASFV vaccine strains, which were initially developed and grown in primary swine macrophage cell cultures, signifying a restriction in the manufacture of a vaccine with commercial purposes.

Pathogens 2024 , 9 Figure 1 .
Figure 1.Virus yields of ASFV-G-Δ9GL/ΔUK in sequential passages in IPKM cells.ASFV-G-Δ9GL/ΔUK and ASFV g were sequentially passed 10 times (MOI = 1) in IPKM cell cultures.Viral titers in each passage were evaluated in primary swine macrophages and values expressed as HAD50/mL.Data represent averages and SD of two experiments.

Figure 1 .
Figure 1.Virus yields of ASFV-G-∆9GL/∆UK in sequential passages in IPKM cells.ASFV-G-∆9GL/∆UK and ASFV g were sequentially passed 10 times (MOI = 1) in IPKM cell cultures.Viral titers in each passage were evaluated in primary swine macrophages and values expressed as HAD 50 /mL.Data represent averages and SD of two experiments.

5 of 9 9 Figure 2 .
Figure 2.In vitro growth kinetics of ASFV-G-Δ9GL/ΔUkp10 in primary swine macrophages (MOI = 0.01).Samples were taken at the indicated time points and titrated in swine macrophages.Titrations were performed in swine macrophages.Data represent means and standard deviations from two independent experiments.The sensitivity of virus detection is ≥log10 1.8 HAD50/mL.The symbol (*) indicates significant differences between ASFV-G-Δ9GL/ΔUKp0 and ASFV-G-Δ9GL/ΔUKp10 at specific time points.Differences were inferred by the unpaired t test using the two-stage set up (Benjamine, Krieger and Yekutieli) method.The reliability of multiple comparisons was evaluated by the false discovery rate method (FDR), considering a q-value < 0.05.

Figure 2 .
Figure 2.In vitro growth kinetics of ASFV-G-∆9GL/∆Ukp10 in primary swine macrophages (MOI = 0.01).Samples were taken at the indicated time points and titrated in swine macrophages.Titrations were performed in swine macrophages.Data represent means and standard deviations from two independent experiments.The sensitivity of virus detection is ≥log10 1.8 HAD 50 /mL.The symbol (*) indicates significant differences between ASFV-G-∆9GL/∆UKp0 and ASFV-G-∆9GL/∆UKp10 at specific time points.Differences were inferred by the unpaired t test using the two-stage set up (Benjamine, Krieger and Yekutieli) method.The reliability of multiple comparisons was evaluated by the false discovery rate method (FDR), considering a q-value < 0.05.