A Cell-Adapted Live-Attenuated Vaccine Candidate Protects Pigs against the Homologous Strain VNUA-ASFV-05L1, a Representative Strain of the Contemporary Pandemic African Swine Fever Virus

African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. Currently, there is no ASF vaccine commercially available. All infected animals must be isolated and culled immediately upon the confirmation of the presence of the virus. Studies leading to the rational development of protective ASF vaccines are urgently needed. Here, we generated a safe and efficacious live-attenuated vaccine (LAV) VNUA-ASFV-LAVL2 by serially passaging a field isolate (VNUA-ASFV-05L1, genotype II) in porcine alveolar macrophages (PAMs, 65 passages) and an immortalized porcine alveolar macrophage cell line (3D4/21, 55 passages). VNUA-ASFV-LAVL2 can efficiently replicate in both PAMs and 3D4/21 cells. It provides 100% protection, even with the low dose of 102 HAD50, to the vaccinated pigs against the challenge of contemporary pandemic ASFV field isolate. Pigs vaccinated with this LAV in a dose range of 102 to 105 HAD50 remained clinically healthy during both the 28-day observation period of immunization and the 28-day observation period of challenge. VNUA-ASFV-LAVL2 was eliminated from blood by 28 days post-inoculation (DPI), and from feces or oral fluids by 17 DPI. Although the vaccine strain in serum remained a safe and attenuated phenotype after five passages in swine, a reversion-to-virulence study using blood or tissue homogenates at peak viremia will be conducted in the future. ASFV-specific IgG antibodies and significant cellular immunity were detected in vaccinated pigs before the ASFV challenge. These results indicate that the VNUA-ASFV-LAVL2 strain is a safe and efficacious LAV against the genotype II ASFV strain responsible for current ASF outbreaks in Asia.


Introduction
African swine fever (ASF) is a highly contagious and severe hemorrhagic transboundary swine viral disease with up to 100% mortality rate, leading to a tremendous socioeconomic loss worldwide [1].The causative agent, ASF virus (ASFV), classified under the Asfarviridae family and Asfivirus genus, is a large, enveloped virus containing a doublestranded DNA (dsDNA) genome of approximately 170-190 kilobase pairs (kbp) [2].A total of 24 ASFV genotypes (I-XXIV) have been described based on the ASFV p72 major capsid protein gene (B646L) [3].The highly virulent ASFV genotype II that emerged in the Caucasus region in 2007 is responsible for the contemporary pandemic in Europe/Asia, and the outbreaks in Caribbean countries (Dominican Republic and Haiti) [4][5][6][7].The first outbreak of ASF in Vietnam was reported in early 2019 and quickly spread across the entire country with more than 8 million piglets depopulated, which was equal to nearly 25 percent of the total pig population in 2020 [8][9][10].At present, the ASFV genotype II has become endemic, and the ASF outbreak is continuing to occur frequently in Vietnam, raising the greatest concerns not only for the government but also for the pig industries.
The clinical presentation and the gross pathological lesions of ASF in domestic pigs may vary depending on the virulence of the virus isolate, the route, the dose of infection, and host characteristics.For acute ASF, the clinical course is characterized by high fever, with temperatures up to 42 • C, lethargy, anorexia, and inactivity [11].The lack of safe and efficacious ASF vaccines is the greatest challenge in the prevention and control of ASF.In the past several years, extensive efforts have been taken to develop ASF vaccines that include inactivated vaccines, recombinant subunit vaccines (protein-based, DNA, viral-vectored), and live-attenuated strains (LAVs) [11][12][13][14].Up to now, the inactivated and recombinant subunit vaccines have not yet been shown to be efficacious [12,[15][16][17].In contrast, recent promising results with LAVs provide hope for a safe and efficacious vaccine against ASF.Several groups have developed LAVs by the deletion of genes associated with virulence, which induce solid protective immunity against homologous strains.Among them, ASFV-G-∆I177L, ASFV-G-∆I177L∆LVR, and ASFV-G-∆MGF strains proved to be an attenuated phenotype in pigs and conferred full protection against the challenge of the parental ASFV-Georgia strain [18][19][20][21][22].However, ideal LAVs that meet the commercial vaccine demands still face challenges related to stable cell lines for producing the LAV at a large scale, and their efficacy depends on the age of pigs, reversion-to-virulence, vaccine virus shedding, and differentiation of infected from vaccinated animals (DIVA) [13,14,23,24].
LAVs, created by passaging a virus in cultured cells, have proven to be an effective means for preventing many viral diseases, including smallpox, polio, measles, mumps, and yellow fever.Those attenuated vaccines elicit strong immunoprotective cellular and antibody responses, and often confer lifelong immunity with only one or two doses [25].However, there is currently no report of using this technology to develop ASF LAVs.Since the identification of ASF outbreak in Vietnam, research towards vaccine development using field ASFV genotype II isolate has been initiated in our groups.Here, we report the generation of a safe and efficacious LAV vaccine VNUA-ASFV-LAVL2 from a field isolate using cell passage.VNUA-ASFV-LAVL2 can not only provide 100% protection to pigs against virulent contemporary pandemic ASFV infection, but also it can efficiently replicate in the commercially available 3D4/21 cell line.

Animals
Piglets (4-7 weeks old) that tested negative for ASFV and ASFV-antibody were obtained from clean pig farms and used for experiments in this study.The pigs were fed a standard commercial diet.In Vietnam, the pigs were housed in the Animal Biosafety Research Facility of the Faculty of Veterinary Medicine, Vietnam National University of Agriculture (VNUA).In the United States, the pigs were housed under laboratory biosafety level III agriculture (BSL3-Ag) conditions at the Biosecurity Research Institute (BRI), Kansas State University (KSU).Animal care and protocols were approved by Institutional Animal

Cells and Virus
Primary pulmonary alveolar macrophages (PAMs) were prepared as described previously [26].PAMs were maintained in a medium containing Dulbecco's modified Eagle medium (DMEM, Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Scientific, Waltham, MA, USA) and Antimycotic (Life Technologies, Grand Island, NY, USA) at 37 • C in 5% CO 2 incubator.
Virulent VNUA-ASFV-05L1 strain (genotype II) was isolated from the spleen of a domestic pig with typical acute ASF during an ASF outbreak in Northern Vietnam in 2020 [27].It is maintained in BSL-3 laboratories of Vietnam National University and Kansas State University.This virus was used for the generation of LAV and the challenge studies conducted on pigs in this study.

In Vitro Evaluation of Virus Passage and Virus Replication
Wild-type virulent VNUA-ASFV-05L1 strain was passaged in PAM cells and 3D4/21 cells.We used the same culture medium for infection and passage of VNUA-ASFV-05L1 strain in PAM cells.For 3D4/21 cells, we added 1.25% dimethyl sulfoxide (DMSO) in the culture medium for improving its ability to support VNUA-ASFV-05L1 replication.Monolayers were infected with VNUA-ASFV-05L1 at the multiplicity of infection (MOI) of 1 and incubated for 4 days.Culture supernatant was then harvested, titrated, and passaged onto fresh monolayers at MOI of 1.The VNUA-ASFV-05L1 was passaged 65 times in PAM cells first and then 55 times in 3D4/21 cells to generate VNUA-ASFV-LAVL2.Virus titers in the supernatants at each passage were titrated in PAMs.PAMs were pre-seeded (80-100% confluent) and incubated with 10-fold dilutions of the harvested supernatants.After 2 h incubation, 2% porcine red blood cells were added for hemadsorption (HAD) testing.After four days culture, the presence of ASFV was assessed using HAD under an inverted microscope.HAD 50 was calculated using the method of Reed and Muench [28].
For testing the in vitro replication characteristics of VNUA-ASFV-LAVL2 and its parental virus (VNUA-ASFV-05L1), monolayers of PAMs and 3D4/21 cells at 90% confluency in 24-well culture plates were infected with the viruses at MOI of 1.After 2 h incubation, the inoculum was removed.Cells were washed and replaced with fresh culture media.Cultures (including cells and culture medium) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 h post-infection (HPI).The collected cultures were subjected to three freeze-thaw cycles.After centrifuging the cell debris, virus titers in the supernatant were tested and calculated as described above.All experiments were performed in duplicate.
To further evaluate the safety of the LAV, we pooled the serum of pigs inoculated with 10 3 HAD 50 /dose of VNUA-ASFV-LAVL2, and we i.m. inoculated 1 mL/dose in pigs (n = 3) to obtain passage 1 (P1).At 9 DPI, when the serum samples showed the highest Ct value with ASFV real-time PCR (RT-PCR), we pooled the collected serum samples from P1 pigs and i.m. inoculated 1 mL/dose in pigs (n = 3) to obtain P2.Five passages were performed with the same method and procedure.Blood and serum samples were collected at 3, 5, 7, 9, 11, 14, 17, 21, 24, 28, and 35 DPI.Clinical signs (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea, and cough), body temperature, and survival rate were recorded daily throughout the experiment.

Efficacy Evaluation of LAV in Pigs
At 28 DPI, pigs in groups inoculated with 10 2 , 10 3 , 10 4 , and 10 5 HAD 50 /dose of VNUA-ASFV-LAVL2 and in DMEM-inoculated control group were challenged (i.m.) with 1 × 10 3 HAD 50 of parental VNUA-ASFV-05L1.The blood, oral fluids, rectal swabs, and serum samples of pigs were collected at 0, 3,5,7,9,11,14,17,21,25, and 28 days postchallenge (DPC).The presence of clinical signs, body temperature, and survival rate were recorded daily throughout the experiment.The dead pigs in the control group were assessed for typical signs of ASFV pathological lesions.Tissue samples (1.5 g) from inner part of brain, kidney, liver, spleen, lung, heart, lymph nodes, stomach, small intestine, large intestine, and bone marrow were aseptically collected from all dead pigs during the experiment, and pigs were euthanized at 28 DPC by qualified personnel.

DNA Extraction, Quantitative ASFV Real-Time PCR, and Genome Sequencing
Quantitative RT-PCR was conducted to detect ASFV DNA in oral fluid and rectal swabs, blood, and tissue homogenates of the experimental pigs.DNA was extracted using an automated King Fisher TM Duo Prime DNA/RNA extraction system (Thermo Fisher Scientific, Waltham, MA, USA) with MagMAX CORE nucleic acid purification kit (Life Sciences, New York, NY, USA), according to the manufacturer's protocols.ASFV DNA was then detected using Platinum SuperMix-UDG kit (Invitrogen, Waltham, MA, USA) on CFX Optus 96 Real-time PCR system (BioRad, Hercules, CA, USA) using p72 primers and the probe developed by Haines et al. [29].Samples with Ct values <40 were considered to be positive.

ASFV-Specific Antibody Detection
The ASF blocking ELISA kit (INGEZIM PPA COMPAC 11.PPA.k3,Ingenasa, Madrid, Spain) was used to detect specific anti-ASFV antibodies in serum samples.The procedure of the commercial ELISA kit was performed as described in the manufacturer's instructions.For each sample, the competition percentage (S/N%) was calculated according to the manufacturer's instructions, and more than or equal to (≥) 50% was considered positive, between 40 and 50% was considered doubtful, and ≤40% was considered negative.
The concentration of porcine IL-10 in serum samples of pigs was measured using porcine IL-10 Quantikine ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).The data from assays for virus titration in cell cultures and blood samples in experimental piglets at different time points and efficacy studies were expressed as the mean log HAD ± SD (Standard deviation) for each group and analyzed with Student's t-test.The data for antibody and cellular responses were expressed as mean readings ± SD for each group.The significance of differences between the experimental groups was analyzed with analysis of variance (ANOVA) followed by Turkey's post-test.For all statistical analyses, p values less than 0.05 were considered as statistically significant.

VNUA-ASFV-LAVL2 Replicates Stably and Efficiently in Both PAMs and 3D4/21 Cells
We compared the growth characteristics of VNUA-ASFV-LAVL2 with its parental virus VNUA-ASFV-05L1 in PAMs and 3D4/21 cells.VNUA-ASFV-LAVL2 showed growth at moderate titers of around 10 5 HAD 50 /mL for the first 15 passages, and then at moderateto-high titers of around 10 5 to 10 6 HAD 50 /mL in 3D4/21 cells.The virus retained this stable replication phenomenon (with similar growth ability) from passage 110 to passage 120 with the highest titer 10 6 HAD 50 /mL at 72 HPI.Passage 120 showed comparable growth kinetics with less than 0.5 log 10 at each time points post-infection in both PAMs and 3D4/21 cells compared to that of parental VNUA-ASFV-05L1 strain (Figure 1A,B).
Mutations that resulted in amino acid substitutions or protein disruptions are found in the genome of VNUA-ASFV-LAVL2 as well (Table 1).Mutations that resulted in amino acid substitutions or protein disruptions are found in the genome of VNUA-ASFV-LAVL2 as well (Table 1).
VNUA-ASFV-LAVL2-vaccinated pigs showed significantly lower viremia than parental VNUA-ASFV-L1-inoculated control pigs.The control pigs reached peak titers of approximately 10 7 HAD 50 /mL before they died, whereas VNUA-ASFV-LAVL2-vaccinated pigs reached the highest viremia titer of 10 4 to 10 5 HAD 50 /mL at 7 to 11 DPI (peak at 11 DPI for low dose, and at 7 DPI for high dose), and then these titers rapidly declined and almost cleared at 28 DPI (Figure 2B,D,F,H).

VNUA-ASFV-LAVL2 Maintained Safe and Attenuated Phenotype When Passaged in Pigs Determined with Pooled Serum Samples
To further evaluate the safety of VNUA-ASFV-LAVL2, we performed its serial passaging in pigs determined with pooled serum samples, which showed the highest Ct with ASFV RT-PCR.In total, five passages (P1 to P5) in pigs were completed in this study.Pigs in all passages showed transient low fever (<40.6 °C) by 6-9 DPI, then showed normal body temperature during the observation period (Figure 4A), and no obvious ASFV-specific clinical signs were observed.All pigs maintained a good daily feed intake between 2 to 5 pounds and were healthy and showed 100% survival until the end of tests.Viremia study showed that replication of VNUA-ASFV-LAVL2 reached its peak Ct value of about 30 (about 10 4 HAD50/mL) at 9 DPI, and then the Ct value rapidly decreased, and VNUA-ASFV-LAVL2 was absent in blood from 11 DPI to 35 DPI (Figure 4B).All passages showed similar viremia and clearance rate of VNUA-ASFV-LAVL2 in inoculated pigs.

VNUA-ASFV-LAVL2 Maintained Safe and Attenuated Phenotype When Passaged in Pigs Determined with Pooled Serum Samples
To further evaluate the safety of VNUA-ASFV-LAVL2, we performed its serial passaging in pigs determined with pooled serum samples, which showed the highest Ct with ASFV RT-PCR.In total, five passages (P1 to P5) in pigs were completed in this study.Pigs in all passages showed transient low fever (<40.6 • C) by 6-9 DPI, then showed normal body temperature during the observation period (Figure 4A), and no obvious ASFV-specific clinical signs were observed.All pigs maintained a good daily feed intake between 2 to 5 pounds and were healthy and showed 100% survival until the end of tests.Viremia study showed that replication of VNUA-ASFV-LAVL2 reached its peak Ct value of about 30 (about 10 4 HAD 50 /mL) at 9 DPI, and then the Ct value rapidly decreased, and VNUA-ASFV-LAVL2 was absent in blood from 11 DPI to 35 DPI (Figure 4B).All passages showed similar viremia and clearance rate of VNUA-ASFV-LAVL2 in inoculated pigs. detection.

VNUA-ASFV-LAVL2 Maintained Safe and Attenuated Phenotype When Passaged in Pig Determined with Pooled Serum Samples
To further evaluate the safety of VNUA-ASFV-LAVL2, we performed its serial p saging in pigs determined with pooled serum samples, which showed the highest Ct w ASFV RT-PCR.In total, five passages (P1 to P5) in pigs were completed in this study.P in all passages showed transient low fever (<40.6 °C) by 6-9 DPI, then showed nor body temperature during the observation period (Figure 4A), and no obvious ASFV-s cific clinical signs were observed.All pigs maintained a good daily feed intake betwee to 5 pounds and were healthy and showed 100% survival until the end of tests.Vire study showed that replication of VNUA-ASFV-LAVL2 reached its peak Ct value of ab 30 (about 10 4 HAD50/mL) at 9 DPI, and then the Ct value rapidly decreased, and VNU ASFV-LAVL2 was absent in blood from 11 DPI to 35 DPI (Figure 4B).All passages show similar viremia and clearance rate of VNUA-ASFV-LAVL2 in inoculated pigs.To test the efficacy of VNUA-ASFV-LAVL2, pigs in both vaccinated groups and the control group (inoculated with DMEM) were challenged with parental VNUA-ASFV-05L1, the contemporary pandemic genotype II ASFV.After challenge, pigs in the control group rapidly displayed clinical signs of ASF (from 5 to 9 DPC), including high fever (Figure 5A), anorexia, cough, depression, staggering gait, and diarrhea, and died from 8 to 9 DPC.In contrast, pigs vaccinated with VNUA-ASFV-LAVL2 (from low dose of 10 2 HAD 50 to high dose of 10 5 HAD 50 ) did not show elevated body temperatures after challenge (Figure 5A).The vaccinated pigs exhibited a high level of protection with 100% survival and were found to be healthy (without clinical signs) when challenged with virulent VNUA-ASFV-05L1.Remarkably, only a very low level of ASFV was detected in the blood before 5 DPC (Figure 5B), and ASFV was not detected in oral fluid or rectal swabs of all vaccinated groups.However, in the control group, the ASFV was detected in blood samples from 3 to 5 DPC, and it rapidly increased from 5 to 9 DPC (Figure 5B).
To further test the protective efficacy of vaccination, we used a higher challenge dose (8 × 10 3 HAD50) than the standard challenge dose for the VNUA-ASFV-LAVL2-vaccinated pigs (10 2 and 10 3 HAD50/dose).In addition, we housed two contact control pigs of each pig group in the same cage.The results showed that all vaccinated groups induced full protection, with 100% survival, and all pigs in those groups were found to be healthy when challenged with high doses of virulent VNUA-ASFV-05L1 strain (Figure 5C).Almost no ASFV was detected in blood (Figure 5D), oral fluid, and rectal swab samples of vaccinated pigs during the 28-day period of challenge.However, ASFV was detected in blood samples of control pigs and contact control pigs at 3 DPC, and it reached its highest titer from 7 to 9 DPC.The pigs from control group and contact control group died between 7 to 9 DPC.To further test the protective efficacy of vaccination, we used a higher challenge dose (8 × 10 3 HAD 50 ) than the standard challenge dose for the VNUA-ASFV-LAVL2-vaccinated pigs (10 2 and 10 3 HAD 50 /dose).In addition, we housed two contact control pigs of each pig group in the same cage.The results showed that all vaccinated groups induced full protection, with 100% survival, and all pigs in those groups were found to be healthy when challenged with high doses of virulent VNUA-ASFV-05L1 strain (Figure 5C).Almost no ASFV was detected in blood (Figure 5D), oral fluid, and rectal swab samples of vaccinated pigs during the 28-day period of challenge.However, ASFV was detected in blood samples of control pigs and contact control pigs at 3 DPC, and it reached its highest titer from 7 to 9 DPC.The pigs from control group and contact control group died between 7 to 9 DPC.
spot-forming cells from VNUA-ASFV-LAVL2-vaccinated pigs were significantly 0.0001) higher than the cells from non-vaccinated pigs (Figure 8A).Cytokine IL-10 EL analysis showed that vaccination with VNUA-ASFV-LAVL2 induced the expression o 10 in pig serum, and it reached its peak at 7 DPI.However, after challenge with the w type VNUA-ASFV-05L1, non-vaccinated control pigs showed significantly higher se IL-10 levels than those of pigs vaccinated with VNUA-ASFV-LAVL2, and these le reached their peak at 7 DPC (35 DPI) (Figure 8B).

Discussion
With the aim of developing a safe and efficacious ASF vaccine, we attempted to erate LAVs using cell passage.3D4/21 cell line is derived from a single cell clone of parent (a continuous porcine alveolar macrophages cell line).It has been reported 3D4/21 can support the growth of cell-adapted ASFV-Lisbon 61 and field isolate L SI/85 [31,32], but it was unable to maintain replication of the genotype II ASFV-HL strain [33].However, a more recent study showed that the ASFV genotype II s CN/GS/2018 can attach and enter 3D4/21 cells, resulting in genome replication rate o to 10 6 copies/mL [34].Our result is consistent with this study.The cell-adapted geno II ASFVs can infect and replicate efficiently in 3D4/21 with a final concentration of 1 DMSO in the culture medium.We have developed several different LAV candidates u this technology and tested their safety in experimental pigs.
VNUA-ASFV-LAVL2, derived from the field isolate (VNUA-ASFV-05L1, geno II), showed one of the best attenuated phenotypes, and the best ability to induce prote immunity in pigs.Whole genome sequencing and analysis at passage 120 showed VNUA-ASFV-LAVL2 harbored d 13 known gene deletions and 14 uncharacterized quence deletions in the MGF region of VNUA-ASFV-LAVL2 (Table 1).VNUA-AS LAVL2 can efficiently grow in both PAMs and 3D4/21 cells, and it showed compar growth kinetics with less than 0.5 log10 at each time point post-infection compared to

Discussion
With the aim of developing a safe and efficacious ASF vaccine, we attempted to generate LAVs using cell passage.3D4/21 cell line is derived from a single cell clone of 3D4 parent (a continuous porcine alveolar macrophages cell line).It has been reported that 3D4/21 can support the growth of cell-adapted ASFV-Lisbon 61 and field isolate Lillie SI/85 [31,32], but it was unable to maintain replication of the genotype II ASFV-HLJ/18 strain [33].However, a more recent study showed that the ASFV genotype II strain CN/GS/2018 can attach and enter 3D4/21 cells, resulting in genome replication rate of up to 10 6 copies/mL [34].Our result is consistent with this study.The cell-adapted genotype II ASFVs can infect and replicate efficiently in 3D4/21 with a final concentration of 1.25% DMSO in the culture medium.We have developed several different LAV candidates using this technology and tested their safety in experimental pigs.
VNUA-ASFV-LAVL2, derived from the field isolate (VNUA-ASFV-05L1, genotype II), showed one of the best attenuated phenotypes, and the best ability to induce protective immunity in pigs.Whole genome sequencing and analysis at passage 120 showed that VNUA-ASFV-LAVL2 harbored d 13 known gene deletions and 14 uncharacterized sequence deletions in the MGF region of VNUA-ASFV-LAVL2 (Table 1).VNUA-ASFV-LAVL2 can efficiently grow in both PAMs and 3D4/21 cells, and it showed comparable growth kinetics with less than 0.5 log 10 at each time point post-infection compared to that of parental VNUA-ASFV-05L1 strain (Figure 1).This phenomenon indicates that the deleted known genes and uncharacterized sequences play a less crucial role in the ASFV replication.Mutations or deletions in the MGF region have been linked to the replication ability and the virulence attenuation of ASFV in both cell culture and pigs [19][20][21]35,36].Therefore, it is not surprising that the deletion of these known MGF genes may play an important role in the attenuated phenotype of VNUA-ASFV-LAVL2 in experimental pigs.However, the replication and attenuation of VNUA-ASFV-LAVL2 in 3D4/21 cells may be associated with other deletions or mutations in the whole genome under selective pressures during the passaging of cells.To clarify the molecular mechanisms involved in the replication and attenuation of VNUA-ASFV-LAVL2, further investigations on the comparison of the whole genome sequences of different passages of VNUA-ASFV-LAVL2 in 3D4/21 cells and their virulence in pigs are needed.
A variety of strategies have been described for developing ASFV LAVs that are able to persist for an extended period in host blood and tissues following immunization, and a correlation in terms of protection was found between enhanced clearance of virulent ASFVs and persistence of LAVs [12,13,23].However, a potential drawback of using these LAVs in animals is the possibility of incomplete clearance of the vaccine strains that could result in reversion-to-virulence.Our findings from this study were particularly encouraging Viruses 2023, 15, 2089 6 of 16with the highest titer 10 6 HAD50/mL at 72 HPI.Passage 120 showed comparable growth kinetics with less than 0.5 log10 at each time points post-infection in both PAMs and 3D4/21 cells compared to that of parental VNUA-ASFV-05L1 strain (Figure1A,B).

Figure 3 .
Figure 3. Viral titers in (A) oral fluids swabs and (B) rectal swabs of VNUA-ASFV-LAVL2-vaccinated pigs and parental VNUA-ASFV-05L1-inoculated control pigs.Data are presented as mean ± SD for pigs in each group.The dotted line represents the limit of detection.

Figure 4 .
Figure 4. Safety of VNUA-ASFV-LAVL2 strain when passaged in pigs.(A) shows rectal temperatures of pigs from P2 to P5. (B) shows Ct values of ASFV in blood samples of pigs from P2 to P5.Data are presented as mean ± SD for pigs in each group.

Figure 3 .
Figure 3. Viral titers in (A) oral fluids swabs and (B) rectal swabs of VNUA-ASFV-LAVL2-vaccinated pigs and parental VNUA-ASFV-05L1-inoculated control pigs.Data are presented as mean ± SD for pigs in each group.The dotted line represents the limit of detection.

Figure 3 .
Figure 3. Viral titers in (A) oral fluids swabs and (B) rectal swabs of VNUA-ASFV-LAVL2-vaccin pigs and parental VNUA-ASFV-05L1-inoculated control pigs.Data are presented as mean ± SD pigs in each group.The dotted line represents the limit of detection.

Figure 4 .
Figure 4. Safety of VNUA-ASFV-LAVL2 strain when passaged in pigs.(A) shows rectal temp tures of pigs from P2 to P5. (B) shows Ct values of ASFV in blood samples of pigs from P2 to Data are presented as mean ± SD for pigs in each group.

Figure 4 .
Figure 4. Safety of VNUA-ASFV-LAVL2 strain when passaged in pigs.(A) shows rectal temperatures of pigs from P2 to P5. (B) shows Ct values of ASFV in blood samples of pigs from P2 to P5.Data are presented as mean ± SD for pigs in each group.

Figure 5 .
Figure 5. Rectal temperature and viremia of pigs after being challenged with virulent VNUA-ASFV-05L1 genotype II strain.(A) Rectal temperature of vaccinated and non-vaccinated control pigs after being challenged with standard dose of virulent VNUA-ASFV-05L1 strain.(B) Viremia of vaccinated and non-vaccinated control pigs after being challenged with standard dose of virulent VNUA-ASFV-05L1 strain.(C) Rectal temperature of vaccinated, non-vaccinated, and contact pigs after being challenged with a high dose of virulent VNUA-ASFV-05L1 strain.(D) Viremia of

Figure 5 .
Figure 5. Rectal temperature and viremia of pigs after being challenged with virulent VNUA-ASFV-05L1 genotype II strain.(A) Rectal temperature of vaccinated and non-vaccinated control pigs after being challenged with standard dose of virulent VNUA-ASFV-05L1 strain.(B) Viremia of vaccinated and non-vaccinated control pigs after being challenged with standard dose of virulent VNUA-ASFV-05L1 strain.(C) Rectal temperature of vaccinated, non-vaccinated, and contact pigs after being challenged with a high dose of virulent VNUA-ASFV-05L1 strain.(D) Viremia of vaccinated, nonvaccinated, and contact pigs after being challenged with a high dose of virulent VNUA-ASFV-05L1 strain.The dotted line represents the limit of detection.
Care and Use Committee at Vietnam National University of Agriculture (VNUA-2021/01) and at Kansas State University (IACUC#4845).All animal experiments were conducted strictly adhering to the IACUC protocols.