Deletion of the A137R Gene from the Pandemic Strain of African Swine Fever Virus Attenuates the Strain and Offers Protection against the Virulent Pandemic Virus

ABSTRACT

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine within an extended geographical area from Central Europe to East Asia, resulting in economic losses for the regional swine industry. There are no commercial vaccines; therefore, disease control relies on identification and culling of infected animals. We report here that the deletion of the ASFV gene A137R from the highly virulent ASFV-Georgia2010 (ASFV-G) isolate induces a significant attenuation of virus virulence in swine. A recombinant virus lacking the A137R gene, ASFV-G-ΔA137R, was developed to assess the role of this gene in ASFV virulence in domestic swine. Animals inoculated intramuscularly with 10² 50% hemadsorption doses (HAD₅₀) of ASFV-G-ΔA137R remained clinically healthy during the 28-day observational period. All animals inoculated with ASFV-G-ΔA137R had medium to high viremia titers and developed a strong virus-specific antibody response. Importantly, all ASFV-G-ΔA137R-inoculated animals were protected when challenged with the virulent parental strain ASFV-G. No evidence of replication of challenge virus was observed in the ASFV-G-ΔA137R-inoculated animals. Therefore, ASFV-G-ΔA137R is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce protection against the highly virulent ASFV Georgia virus that is the cause of the current Eurasian pandemic.

IMPORTANCE No commercial vaccine is available to prevent African swine fever. The ASF pandemic caused by ASFV Georgia2007 strain (ASFV-G) is seriously affecting pork production in a contiguous area from Central Europe to East Asia. Here we report the rational development of a potential live attenuated vaccine strain by deleting a virus-specific gene, A137R, from the genome of ASFV-G. The resulting virus presented a completely attenuated phenotype and, importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. ASFV-G-ΔA137R confers protection even at low doses (10² HAD₅₀), demonstrating its potential as a vaccine candidate. Therefore, ASFV-G-ΔA137R is a novel experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

INTRODUCTION

African swine fever virus (ASFV) causes a highly contagious disease of domestic swine, African swine fever (ASF). ASF is endemic in more than 20 sub-Saharan African countries and, since its eruption in the Caucasus region in 2007, has rapidly spread, currently affecting countries in a contiguous geographical area from Central Europe to East and Southeast Asia. This current pandemic is causing alarming economic losses in the pig industry and a shortage in protein availability in the affected countries. The ASFV causing the pandemic is a highly virulent isolate that belongs to the ASFV genotype II group (1) and has remained genetically stable with only minor genome modifications compared to the initial 2007 outbreak strain, ASFV Georgia 2007/1.

ASFV is a complex, large, and enveloped virus harboring a double-stranded DNA (dsDNA) genome of approximately 180 to 190 kbp (2) which encodes approximately 150 to 160 open reading frames (ORFs). The functions of many of the ASFV proteins remain unknown or have been predicted by functional genomics, with only a few proteins undergoing experimental evaluation studies to determine their function or host protein binding partners (3–11). The deletion of single genes in ASFV often results in no phenotypic change to the virus pathogenesis (3, 12–17).

No vaccine is available for ASF, therefore disease control is managed using quarantine and culling of affected animals. Pigs surviving viral infection or infected with natural attenuated variants of ASFV develop long-term protection against reinfection with the homologous virulent virus (2). Pigs immunized with live attenuated ASF viruses derived from the pandemic ASFV strain backbone containing genetically engineered deletions of specific ASFV virulence-associated genes are protected when challenged with homologous parental virus (18–25). Another ASFV backbone, Badajoz71, has also been used with genetic deletions with protection against the current pandemic strain (26). These reports constitute the only evidence describing the rational development of effective experimental live attenuated vaccines (LAVs) against the current pandemic strain of ASFV. Therefore, the identification of virus genes involved in the process of virus virulence is a critical initial step in the development of recombinant LAVs to control ASFV infection and disease.

As first generation LAVs against ASFV have shown some successes, additional genetic deletions may be required to increase their safety profile. However, the combination of multiple gene deletions has been met with challenges, since combining multiple genetic deletions often has resulted in decreased virus replication in swine and reduced protective efficacy (27–29). In addition, deletion of even highly conserved genes may have different effects on viral virulence, depending on the virus isolate considered (26, 30). Therefore, the identification and characterization of novel genetic determinants of virulence is of critical importance in the rational development of the next-generation LAVs against the current pandemic strain ASFV. That information may be used to increase the safety of current vaccine candidates, as well as to achieve attenuation in new emerging strains of ASFV.

Here, we report the discovery that deletion of the ASFV gene A137R from the highly virulent ASFV Georgia isolate ASFV-G results in virus attenuation in swine. Animals inoculated with a recombinant virus lacking the A137R gene, ASFV-G-ΔA137R, remained clinically normal, developed a strong virus-specific antibody response, and are completely protected when challenged with the virulent parental ASFV-G.

RESULTS

Conservation of A137R gene across different ASFV isolates.

As originally described (31), the ASFV A137R gene encodes a 137-amino acid protein and is positioned on the positive strand between nucleotide positions 55531 and 55944 of the ASFV-G genome (Fig. 1). The translated product of the ASFV A137R gene is a protein expressed late during the virus replication cycle, with an electrophoretic mobility of 11.5 kDa, and has been detected in preparations of purified virus particles and in virus factories (31, 32).

FIG 1.

Diagram indicating the position of the A137R open reading frame in the ASFV-G genome. The donor plasmid with the homologous arms to ASFV-G and the mCherry gene under the control of the p72 promoter in the orientation as indicated. The final genomic changes introduced to develop ASFV-G-ΔA137R were where the sequence of the donor plasmid mCherry reporter was introduced to replace the ORF of A137R as indicated (bottom). The nucleotide positions indicated refer to the nucleotide positions in the parental ASFV-G genome.

Evaluation of the conservation of the A137R gene at the nucleotide and amino acid levels among different ASFV isolates was performed using the CLUSTALW algorithm (33). Overall, the average homology at nucleotide and amino acid levels were calculated to be 93.34% and 90.39%, respectively. However, there is a disparate range of homology at nucleotide (87.5 to 100%) and amino acid (82.35 to 100%) levels, indicating a moderate conservation of the A137R protein among ASFV isolates (Fig. 2). Overall, no differences at nucleotide and amino acid levels were found with isolated belonging to the epidemic Eurasia lineage II.

FIG 2.

Multiple sequence alignment of the indicated ASFV isolates of viral protein A137R. Seventeen protein sequences representing the genetic diversity of gene A137R of ASFV at the GenBank database were used to conduct this alignment. Based on pairwise and neighbor joining cluster analyses (conducted on Mega version 10.0.5), isolates were classified into five different groups. Identity between different groups is shown. The alignment contains 104 conserved and 32 variable sites. Among variable sites, conservation scores are displayed based on the biological properties of each amino acid, where the lower scores are associated with more divergent replacements. An asterisk (*) symbol indicates residue conservation, while a plus sign (+) indicates replacement with an amino acid having similar properties. Additionally, black and red arrows represent relevant residues evolving under negative or positive selection, respectively. Alignment was conducted on the Jalview software version 2.11.1.3, using the Clustal W algorithm.

More information about the disparate range of identity at the amino acid level was obtained by performing specific pairwise calculations between isolates using the model p-distance and the bootstrap method (1,000 repetitions) to set the confidence interval at 95%. Based on this approach, and supported by neighbor joining cluster analysis, we congregated different isolates into five groups with an average identity within groups of between 0 and 96.5%. When comparing the identity between groups, we observed that the moderate conservation of this protein was associated with the presence of isolates from group 5, where this group showed an identity with the other groups of between 82.79 and 85.81% (Fig. 2).

No homology was found among 19, 175 protein families when the A137R protein was evaluated using the program Pfam 34.0 (34). Interestingly, evolutionary analysis conducted using the algorithms fixed effects likelihood (FEL) (35) and mixed effects model of evolution (MEME) (36) revealed that negative selection is dominating the evolution of the A137R gene (Ka/K_s = 0.460). A total of nine residues in the protein appeared to be evolving under negative selection (P = 0.1) at positions 22, 23, 56, 59, 83, 95, and 131, reflecting the importance of the conservation of these sites during the evolution of A137R. Conversely, four residues at positions 37, 40, 69, and 93 were found to be evolving under positive selection (P = 0.1), suggesting the importance of these residues during the divergence of A137R protein. No evidence of recombination was found using the genetic algorithm for recombination detection (GARD) (37).

Development of the A137R gene deletion mutant of the ASFV-Georgia isolate.

The function of the A137R gene, which encodes a late-expressed ASFV structural protein, remains completely unknown. To investigate the impact of the function of the A137R gene during ASFV infection in cell cultures and, more importantly, in replication and virulence in swine, a recombinant virus lacking the A137R gene was designed (ASFV-G-ΔA137R). ASFV-G-ΔA137R was developed from the highly virulent parental ASFV Georgia 2010 (ASFV-G) strain through homologous recombination, as described in detail in the Material and Methods. The A137R gene was substituted by a cassette comprising the fluorescent reporter gene mCherry under the ASFV p72 promoter (Fig. 1). The recombinant ASFV-G-ΔA137R was obtained after 14 steps of purification by limiting dilution based on the presence of fluorescent activity. The ASFV-G-ΔA137R population obtained from the last purification step was further amplified in primary swine macrophage cell cultures to develop the virus stock.

Next-generation sequencing (NGS) was used to assess the precision of the genetic modification introduced into ASFV-G-ΔA137R, evaluate the integrity of the genome, and confirm purity of the recombinant virus stock. The comparison of DNA sequence assemblies of ASFV-G-ΔA137R and ASFV-G demonstrated a deletion of 249 nucleotides (covering nucleotide positions 55531 to 55944) from the A137R gene, corresponding with the introduced modification. In addition, the ASFV-G-ΔA137R genome harbors a 3,944-nucleotide insertion corresponding to the p72mCherryΔA137R cassette introduced by replacing the 249-nucleotide deletion of the A137R gene. No undesired additional genomic alterations were observed in the rest of the ASFV-G-ΔA137R genome.

Replication of ASFV-G-ΔA137R in primary swine macrophages.

Primary swine macrophage cells, the primary cell targeted by ASFV during infection in swine, were used in vitro to assess the effect of the A137R deletion on the ASFV genome. The growth kinetics of ASFV-G-ΔA137R and the parental ASFV-G were compared in multistep growth curves (Fig. 3). Swine macrophage cultures were infected at a multiplicity of infection (MOI) of 0.01 and samples were collected at 2, 24, 48, 72, and 96 h postinfection (hpi). Results demonstrated that ASFV-G-ΔA137R displayed a growth kinetic significantly decreased when compared to parental ASFV-G. ASFV-G-ΔA137R yields were approximately 10-fold lower than those of ASFV-G at all time points considered between 24 and 96 hpi. Therefore, although not essential for virus replication, deletion of the A137R gene significantly decreased the ability of ASFV-G to replicate in primary swine macrophage cell cultures.

FIG 3.

In vitro growth characteristics of ASFV-G-ΔA137R and parental ASFV-G. Primary swine macrophage cell cultures were infected (MOI = 0.01) with each of the viruses and virus yield was titrated at the indicated times postinfection. Data represent means from three independent experiments. Sensitivity of virus detection: ≥1.8 log₁₀ HAD₅₀/ml. Significant differences (*) in viral yields between both viruses at specific times points were determined using the Holm-Sidak method (α = 0.05) without assuming a consistent standard deviation. All calculations were conducted on the software GraphPad Prism version 8.

Assessment of ASFV-G-ΔA137R virulence in swine.

To assess deletion of the A137R gene in vivo, domestic pigs were infected with ASFV-G-ΔA137R and disease phenotype was compared with animals infected with parental virulent ASFV-G. A group (n = 5) of 80- to 90-pound pigs were intramuscularly (i.m.) inoculated with 10² 50% hemadsorption doses (HAD₅₀) of either ASFV-G-ΔA137R or ASFV-G. A sixth mock-inoculated animal, designated “sentinel,” was kept in cohabitation to detect the potential presence of virus shedding from the inoculated animals. As expected, all animals inoculated with ASFV-G presented with an increase in body temperature (>104°F) by day 5 postinfection. All ASFV-G-inoculated animals presented with ASF clinical signs, including anorexia, depression, purple skin discoloration, staggering gait, and diarrhea. Clinical disease progressively worsened, with all animals euthanized in extremis by 7 days postinfection (pi) (Table 1, Fig. 4, and Fig. 5). Conversely, the five animals inoculated via i.m. with ASFV-G-ΔA137R did not present with any ASF-related signs, remaining clinically normal during the entire 28-day observation period except for the appearance of mild and transient increased body temperature (Fig. 4). Therefore, deletion of the A137R gene produced a dramatic attenuation of the virulent ASFV-G strain.

TABLE 1.

Swine survival and fever response following infection with 10² HAD₅₀ doses of ASFV-G-ΔA137R or parental ASFV-G

Virus inoculant	No. of survivors/ total	Mean time to death (days [SD])	Fever
			No. of days to onset (SD)	No. of days duration (SD)	Maximum daily temp (°F [SD])
ASFV-G	0/5	7 (0)a	4.6 (0.55)	2.4 (0.55)	105.52 (0.79)
ASFV-G-ΔA137R	5/5	NAa	NA	NA	103.4 (0.46)

^aAll animals were euthanized due to humanitarian reasons following the corresponding IACUC protocol. NA, not applicable.

FIG 4.

Kinetics of body temperature values in pigs i.m. inoculated with 10² HAD₅₀ of either ASFV-G-ΔA137R or ASFV-G (ASFV-G 1) before and after the challenge with 10² HAD₅₀ of ASFV-G (ASFV-G 2). Each curve represents an individual animal’s values in each of the groups.

FIG 5.

Kinetics of mortality in pigs i.m. inoculated with 10² HAD₅₀ of either ASFV-G-ΔA137R or ASFV-G (ASFV-G 1) before and after the challenge with 10² HAD₅₀ of ASFV-G (ASFV-G 2).

Viremia kinetics in ASFV-G-infected animals presented, as expected, with high titers (10^5.3 to 10^8.3 HAD₅₀/ml) on day 4 pi. These titers increased (around 10^7.5 to 10^8.5 HAD₅₀/ml) by day 7 pi, when all animals were euthanized (Fig. 6). Conversely, ASFV-G-ΔA137R revealed a different pattern, with lower viremia titers (10^1.8 to 10^5.8 HAD₅₀/ml) at day 4 pi, reaching peak values (approximately 10⁵ to 10⁶ HAD₅₀/ml) by day 11 pi, with titers remaining steady (around 10⁵ HAD₅₀/ml) until day 28 pi in 4 of the 5 animals (Fig. 6). It should be noted that one of the five animals inoculated with ASFV-G-ΔA137R had undetectable viremia levels until 21 dpi, when a viremia titer of 10⁴ HAD₅₀/ml was detected. Therefore, disappearance of ASFV virulence caused by deletion of the A137R gene is accompanied by a reduced but stable virus replication, presenting as long viremias with relatively low titer values.

FIG 6.

Viremia titers detected in pigs i.m. inoculated with 10² HAD₅₀ of either ASFV-G-ΔA137R or ASFV-G (ASFV-G 1) before and after the challenge with 10² HAD₅₀ of ASFV-G (ASFV-G 2). Each curve represents values from individual animals in each group. Sensitivity of virus detection: ≥1.8 log₁₀ HAD₅₀/ml.

Interestingly, while no virus was detected in any of the blood samples obtained (Fig. 6), virus nucleic acid was detected by PCR in tonsils (but not in spleen) obtained at 28 days pi of sentinel animals (Table 2), indicating that ASFV-G-ΔA137R-infected animals did shed enough virus to infect naive pigs during the 28 days of cohabitation.

TABLE 2.

Identification of virus detected at 21 days postchallenge in tonsils and spleens of animals infected with ASFV-G-ΔA137R and challenged with ASFV-G virus

Treatment	Spleena			Tonsilsa
	p72	A137R	mCherry	p72	A137R	mCherry
Infected/challenged	21.8	Neg	22.03	15.11	Neg	16.68
Infected/challenged	27.9	Neg	27.91	15.52	Neg	16.72
Infected/challenged	26.25	Neg	26.51	16.86	Neg	18.12
Infected/challenged	Neg	Neg	Neg	22.3	Neg	22.63
Infected/challenged	28.06	Neg	28.21	16.23	Neg	16.8
Sentinelb	Neg	Neg	Neg	19.72	Neg	20.3

^aReal time PCR were run as described in the Material and Methods to detect p72, A137R, and mCherry genes. Data is presented as C_T (threshold cycle) values. Neg, no signal after 40 cycles of amplification.

^bSentinel’s samples were obtained at day 28 pi.

Protective efficacy of ASFV-G-ΔA137R against challenge with parental ASFV-G.

Animals infected with attenuated ASFV strains, regardless of their origin, usually induce protection against infection or disease caused by the virulent homologous virus (18–25). To evaluate the capability of ASFV-G-ΔA137R infection to protect against challenge with highly virulent parental virus ASFV-G, the animals infected with 10² HAD₅₀ of ASFV-G-ΔA137R were challenged 28 days later with 10² HAD₅₀ of ASFV-G by i.m. route. An additional group (n = 5) of naive animals were included as a mock-inoculated control group and challenged under the same conditions.

Mock animals started showing clinical signs of the disease by 4 to 5 days postchallenge (dpc) and increasing in severity quickly, with all animals euthanized by day 7 pc (Table 3, Fig. 4, and Fig. 5). Conversely, animals in the group infected with ASFV-G-ΔA137R remained clinically normal during the 21-day observation period. Therefore, infection with ASFV-G-ΔA137R induced protection against clinical disease after animals were challenged with the highly virulent parental virus.

TABLE 3.

Swine survival and fever response in ASFV-G-ΔA137R-infected animals challenged with 10² HAD₅₀ ASFV-G virus 28 days later

Virus inoculant	No. of survivors/ total	Mean time to death (days [SD])	Fever
			No. of days to onset (SD)	No. of days duration (SD)	Maximum daily temp (°F [SD])
Mock	0/5	7 (0)a	4.2 (0.45)	2.8 (0.45)	105.98 (0.94)
ASFV-G-ΔA137R	5/5	NAa	NA	NA	102.22 (1.06)

^aAll animals were euthanized due to humanitarian reasons following the corresponding IACUC protocol. NA, not applicable.

As expected, viremia titers in the control animals challenged with ASFV-G were high (ranging between 10^5.8 and 10⁸ HAD₅₀/ml) on day 4 pi, quickly increasing (ranging 10^7.3 to 10^8.8 HAD₅₀/ml) by day 7 pi, when all animals were euthanized. After challenge, none of the animals previously infected with ASFV-G-ΔA137R developed viremia titers with higher values than those present at the time of challenge. Viremia values in all animals progressively decreased until the end of the experimental period (21 days after challenge) when, importantly, no circulating virus could be detected in the blood of four of the five animals in this group (Fig. 6). The remaining animal had very low viremia titers (10^2.8 HAD₅₀/ml) at day 21 pc.

To assess the potential replication of the challenge virus, tonsil and spleen samples obtained from ASFV-G-ΔA137R-infected animals were obtained at the end of the observational period (21 days postchallenge). All samples were then tested using the p72-, A137R-, and mCherry-specific real-time PCRs. As performed, the A137R-specific test was shown to be as sensitive as the one to detect p72 (data not shown). All positive samples detected the presence of p72 and mCherry genes, but were negative for A137R, indicating the absence of challenge virus (Table 2). These results indicate that evidence of challenge virus replication was not observed in tonsils and spleen of ASFV-G-ΔA137R-immunized animals at 21 days pc.

Host antibody response in animals infected with ASFV-G-ΔA137R.

The identification of host immune mechanisms mediating protection against virulent strains of ASFV in animals infected with attenuated strains of virus is under debate. To our knowledge, the only parameter consistently associated with protection against challenge is the presence of virus-specific circulating antibodies. To better understand immune mechanisms induced by infection with ASFV-G-ΔA137R, we tried to associate the presence of anti-ASFV circulating antibodies with protection against challenge. A strong ASFV-specific antibody response was detected in the sera of all these animals using an in-house-developed direct enzyme-linked immunosorbent assay (ELISA) (Fig. 7). Antibody response, mediated by IgG isotypes, was detected in three of the animals by day 11 pi. By day 14 pi, the antibody response reached maximum levels in all animals. Therefore, as described in our previous reports, there is a close correlation between presence of anti-ASFV antibodies at the moment of challenge and protection.

FIG 7.

Anti-ASFV antibody titers detected by ELISA in pigs i.m. inoculated with 10² HAD₅₀ of ASFV-G-ΔA137R and in the sentinel pig. Each point represents values from individual animals.

DISCUSSION

Among all ASFV experimental vaccines tested, attenuated strains are the most credible alternative to produce a successful vaccine. Attenuated strains developed by genetic manipulation, specifically removing virus genes associated with virulence, is currently the more plausible approach to develop a safe and efficacious vaccine protecting against the virulent parental virus (22, 23, 38–41). Here, we describe the discovery of a previously uncharacterized ASFV gene, A137R, that is involved in virus virulence in swine. Removal of the A137R gene attenuates highly virulent ASFV-G in swine. Just a few genes (or gene combinations) have been shown to attenuate virus virulence when deleted from the genome of ASFV-G or its derivatives.

Therefore, deletion of the 9GL gene (alone or potentiated by the additional deletion of the UK gene) (22, 23), the deletion of a group of six genes from the MGF360 and 530 (21), deletion of the I177L gene (19, 20), combined deletion of CD2-like and the UK gene (42), co-deletion of genes L7L, L8, L9L, L10L, and L11L (18), deletion of the MGF110-9L (25), and deletion of the MGF505-7R (24) are the only reports describing attenuation of an ASFV-G strain by deleting specific genes or groups of genes. In most of these cases, recombinant attenuated virus strains developed by genetic manipulation were able to induce protection against challenge with the virulent parental virus, constituting unique examples of experimental vaccines protecting against the field isolate responsible for the current pandemic affecting Eurasia.

Although all animals infected with ASFV-G-ΔA137R strain showed no clinical signs associated with ASF, they have relatively high viremia titers lasting until day 28 pi. Long viremia periods are a common event that occurs in animals infected with attenuated ASFV strains (20, 21, 23). In fact, in our experience, the presence of detectable viremias in animals infected with attenuated virus is strongly associated with protection against the challenge with virulent parental ASFV-G. Therefore, ASFV-G-ΔA137R vigorously replicated after i.m. inoculation and this replication might be associated with the presence of virus shedding, as evidenced by the detection of both the presence of ASFV-G-ΔA137R in tonsils of the sentinel animal as well as the late rise of ASFV-specific antibodies by day 28 pi (Fig. 7).

Animals infected with ASFV-G-ΔA137R were protected when challenged at 28 dpi with the parental virulent ASFV-G, not developing any clinical signs associated with disease (not even a transient rise in body temperature), accentuating the efficacy of protection induced by ASFV-G-ΔA137R, even inoculated at a low dose (10² HAD₅₀). Importantly, it appears that replication of the challenge virus in the ASFV-G-ΔA137R-infected animals is either quite restricted or completely absent, since no challenge virus was isolated in tonsils or spleen of any of the protected animals.

Although the immune responses inducing protection against ASFV are still to be determined, our work using live attenuated vaccine candidates demonstrated a good association between protection and the presence of virus-specific antibodies in serum (19–23). Here, we are again able to detect high levels of ASFV-specific antibodies in all protected animals.

Taken together, the results presented here demonstrate for the first time that ASFV gene A137R is a novel determinant of virulence and that its deletion from the genome of the highly virulent ASFV-G strain produces an attenuated virus strain, ASFV-G-ΔA137R. ASFV-G-ΔA137R, even when administered at a low dose, protected animals against the parental virus, apparently producing a sterile immunity. These results suggest ASFV-G-ΔA137R is a promising vaccine candidate. However, the exact role and function for A137 in ASFV pathogenesis still remains unknown. Functional genomic analysis of the A137R gene did not identify any domains or motifs indicative of particular protein function; further work will need to be done to determine the functional role of A137R.

MATERIALS AND METHODS

Cell culture and viruses.

Primary swine macrophage cell cultures were prepared from heparin-treated swine blood as previously described in detail (43). Briefly, mononuclear leukocytes were separated over a Ficoll-Paque density gradient (Pharmacia, Piscataway, N.J.) and the monocyte/macrophage cell fraction was cultured in plastic Primaria tissue culture flasks (Falcon; Becton, Dickinson Labware, Franklin Lakes, N.J.) containing macrophage medium, composed of RPMI 1640 medium (Life Technologies, Grand Island, NY) with 30% L929 supernatant and 20% fetal bovine serum (HI-FBS, Thermo Fisher Scientific, Waltham, MA) for 48 h at 37°C under 5% CO₂. Adherent cells were detached from the plastic using 10 mM EDTA in phosphate-buffered saline (PBS) and were then reseeded into Primaria T25, 6- or 96-well dishes at a density of 5 × 10⁶ cells per ml for use in assays 24 h later.

Growth curves comparing growth kinetics between ASFV-G and ASFV-G-ΔA137R viruses were performed in primary swine macrophage cell cultures. Macrophage monolayers, prepared in 24-well plates, were infected at an MOI of 0.01 and 1 h later the inoculum was removed, the cells rinsed twice with PBS, once with macrophage medium, and incubated for 2, 24, 48, 72, and 96 h at 37°C under 5% CO₂. At appropriate times postinfection, the cells were frozen at <−70°C and the thawed lysates were used to determine virus titers in primary swine macrophage cell cultures. All samples were run simultaneously to avoid interassay variability.

Virus titration was performed on primary swine macrophage cell cultures in 96-well plates. Virus dilutions and cultures were performed using macrophage medium. Presence of virus was assessed by hemadsorption (HA) and virus titers were calculated by the Reed and Muench method (44).

ASFV Georgia (ASFV-G) was a field isolate kindly provided by Nino Vepkhvadze from the Laboratory of the Ministry of Agriculture (LMA) in Tbilisi, Republic of Georgia. This isolate has the same sequence as GenBank accession number FR682468.2.

Construction of the recombinant ASFV-G-ΔA137R.

Recombinant ASFV-G-ΔA137R was generated by homologous recombination between the parental ASFV-G genome and recombination transfer vector p72mCherryΔA137R by infection and transfection procedures using swine macrophage cell cultures as previously described in detail (45). The recombinant transfer vector p72mCherryΔA137R contained flanking genomic regions to the amino acid residues 1 and 85 of the A137R gene, mapping approximately 1 kbp to the left and right of these amino acids, along with the reporter gene cassette containing the mCherry gene with the ASFV p72 late gene promoter, p72mCherry. This construction created a 249-bp deletion in the A137R ORF, resulting in the deletion of the first 85 amino acids of A137R, such that the remaining coding region is unlikely to be expressed due to the lack of a promoter or start codon. (Fig. 1). The recombinant transfer vector p72mCheryΔA137R was obtained by DNA synthesis (Epoch Life Sciences Missouri City, TX, USA).

Next generation sequencing (NGS) of ASFV genomes.

ASFV DNA was extracted from infected cells and quantified as described earlier. Full-length sequencing of the virus genome was performed as described previously (46) using an Illumina NextSeq500 sequencer.

Animal experiments.

Animal experiments were performed under biosafety level 3AG conditions in the Plum Island Animal Disease Center (PIADC) animal facility following protocols approved by the PIADC Institutional Animal Care and Use Committee of the U.S. Departments of Agriculture and Homeland Security (protocol number 225.04-16-R, approved 09-07-16).

ASFV-G-ΔA137R virulence was assessed in comparison to that of the virulent parental ASFV-G virus using 80- to 90-pound commercial Yorkshire crossbreed swine. Groups of pigs (n = 5) were intramuscularly (i.m.) inoculated with 10² HAD₅₀ of either recombinant ASFV-G-ΔA137R or parental ASFV-G virus. A sixth noninoculated pig was incorporated in the group as sentinel to detect the presence of virus shedding from the inoculated animals. Sentinel cohabitated with the inoculated group until day 28 pi, when it was removed from the room. Presence of clinical signs (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea, and cough) and changes in rectal temperature were recorded daily throughout the experiment. In protection experiments, animals inoculated with ASFV-G-ΔA137R were i.m. challenged 28 days later with 10² HAD₅₀ of parental virulent ASFV-G. The presence of clinical signs associated with the disease was recorded as described earlier (22).

Design of qRT-PCR-A137R.

To rule out the presence of ASFV-G in the blood and organs collected from inoculated pigs with the recombinant ASFV-G-ΔA137R, a quantitative PCR (qPCR) was designed to detect the gene KP-A137R of ASFV. For this purpose, the sequence of the isolate ASFV Georgia 2007/1 (NCBI accession number LR743116) was used as a reference for the design of primers and probe (TaqMan). Sequences for the oligonucleotides are as follows: forward primer 5′-ATTTTCATCGTTGTGCTTGGG-3′; reverse primer 5′-AGGCGGTGTGGAATTCAG-3′; probe FAM-5′-TAGGTGCATCGTTCCTCAGGGATTTC-3′-MGB NFQ. Two additional qPCRs were included as a part of this evaluation. As a positive marker for the presence of ASFV, we used the previously published design for detection of the p72 gene (47), while as a positive marker for the recombinant ASFV-G-ΔA137R, we designed a qRT-PCR to target the gene encoding the fluorescent protein mCherry as follows: forward primer 5′- GCTTCTTGGCCTTGTAGGTG-3′; reverse primer 5′-CAGAGGCTGAAGCTGAAGGA-3′; probe FAM-5′-TAGGTGCATCGTTCCTCAGGGATTTC-3′-MGB NFQ. The sequence of the expression vector precB5R.1 (NCBI accession number LC325569) was used as a reference for this design.

To determine the limit of detection of qRT-PCR-KP-177R, several 10-fold dilutions of a viral stock of ASFV-G with a known titer were prepared using macrophage medium. Each dilution was run in duplicate. As a reference for the comparison, we used the previously published qRT-PCR protocol to detect the p72 gene (47).

Automated DNA extraction was conducted using a KingFisher nucleic acid purification platform and a 5X MagMAX Pathogen RNA/DNA kit (Applied Biosystems catalog 4462359) following the manufacturer’s instructions (50 μl/sample). All qPCR assays were conducted using a 7500 real-time PCR system (Applied Biosystems) and the TaqMan Universal PCR Master Mix (Applied Biosystems catalog 4304437) with the following master mix preparation (1×): universal mix 12.5 μl, water 7.05 μl, forward primer (50 μM) 0.1 μl, reverse primer (50 μM) 0.1 μl, probe (10 μM), and DNA 5 μl. Conditions for amplification were as follows: one cycle at 55°C for 2 min, followed by one denaturalization cycle at 95°C for 10 min, then 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 65°C for 1 min.

Detection of anti-ASFV antibodies.

ASFV antibody detection used an in-house ELISA performed as described previously (48). Briefly, ELISA antigen was prepared from ASFV-infected Vero cells. Maxisorb ELISA plates (Nunc, St. Louis, MO, USA) were coated with 1 μg per well of infected or uninfected cell extract. The plates were blocked with phosphate-buffered saline containing 10% skim milk (Merck, Kenilworth, NJ, USA) and 5% normal goat serum (Sigma, Saint Louis, MO). Each swine serum was tested at multiple dilutions against both infected and uninfected cell antigen. ASFV-specific antibodies in the swine sera were detected using an anti-swine IgG-horseradish peroxidase conjugate (KPL, Gaithersburg, MD, USA) and SureBlue Reserve peroxidase substrate (KPL). Plates were read at an optical density at 630 nm (OD₆₃₀) in an ELx808 plate reader (BioTek, Shoreline, WA, USA). Serum titers were expressed as the log₁₀ of the highest dilution where the OD₆₃₀ reading of the tested sera at least duplicated the reading of the mock-infected serum.