African swine fever virus (ASFV) causes a virulent, deadly infection in wild and domestic swine and is currently causing a pandemic covering a contiguous geographical area from Central and Eastern Europe to Asia. No commercial vaccines are available to prevent African swine fever (ASF), resulting in devastating economic losses to the swine industry. The most advanced vaccine candidates are live attenuated strains developed using a genetically modified virulent parental virus. Recently, we developed a vaccine candidate, ASFV-G-ΔI177L, by deleting the I177L gene from the genome of the highly virulent ASFV pandemic strain Georgia (ASFV-G). ASFV-G-ΔI177L is safe and highly efficacious in challenge studies using parental ASFV-G. Large-scale production of ASFV-G-ΔI177L has been limited because it can replicate efficiently only in primary swine macrophages. Here, we present the development of an ASFV-G-ΔI177L derivative strain, ASFV-G-ΔI177L/ΔLVR, that replicates efficiently in a stable porcine cell line. In challenge studies, ASFV-G-ΔI177L/ΔLVR maintained the same level of attenuation, immunogenic characteristics, and protective efficacy as ASFV-G-ΔI177L. ASFV-G-ΔI177L/ΔLVR is the first rationally designed ASF vaccine candidate that can be used for large-scale commercial vaccine manufacture.
IMPORTANCE African swine fever is currently causing a pandemic resulting in devastating losses to the swine industry. Experimental ASF vaccines rely on the production of vaccine in primary swine macrophages, which are difficult to use for the production of a vaccine on a commercial level. Here, we report a vaccine for ASFV with a deletion in the left variable region (LVR). This deletion allows for growth in stable cell cultures while maintaining the potency and efficacy of the parental vaccine strain. This discovery will allow for the production of an ASF vaccine on a commercial scale.
African swine fever virus (ASFV), the only member of the Asfarviridae family, is a large enveloped virus containing a 180- to 190-kbp double-stranded DNA genome that encodes >150 open reading frames (ORFs) (1). It is the causative agent of African swine fever (ASF), a devastating disease of domestic pigs currently affecting Central and Eastern Europe and Asia (1). Animal losses of >90% during ASF outbreaks have led to significant economic losses and protein shortages on local and global scales (1).
No commercial vaccine is currently available, so animal movement restrictions and culling of infected herds are used to control outbreaks (1). Attenuated experimental vaccines developed using genetic manipulation of virulent isolates contribute to the understanding of the functions of individual virus genes in the process of virus virulence in the natural host. Effective experimental vaccines have been developed by deleting specific virus genes associated with virulence (2–10).
While several experimental vaccine candidates reported to produce solid protection against the highly virulent ASFV pandemic strain Georgia (ASFV-G) or its derivatives have been developed (3–6, 8, 11), a significant hurdle preventing large-scale commercial production is the fact that these viruses can replicate efficiently only in primary swine macrophages, with the exception of strain BA71CD2, which replicates in COS-1 cells (7). Adaptation of ASFV field isolates (or their derivative strains) to replicate in established cell lines has been achieved by successive serial passaging but is usually accompanied by significant modifications to the virus genome (12). These genetic modifications include loss of virus genes and phenotypic changes such as loss of the ability to replicate in swine. For instance, adaptation of ASFV-G to replicate in Vero cells resulted in the deletion of approximately 10 to 15% of its genome and was accompanied by an almost complete loss of replicative ability in swine macrophages and complete attenuation in domestic swine (12).
Recently, we reported the rational development of a live attenuated vaccine candidate, ASFV-G-ΔI177L, obtained by deletion of the I177L gene from the genome of ASFV-G (6). ASFV-G-ΔI177L was shown to be safe even when inoculated parenterally at high doses and to be highly efficacious in inducing protection against challenge with the highly virulent parental strain ASFV-G, even when administered at a relatively low dose. However, commercial production of this attenuated virus is hampered because it replicates exclusively in primary cultures of swine macrophages. Here, we report the development of ASFV-G-ΔI177L/ΔLVR, a derivative strain of ASFV-G-ΔI177L with a deletion in the left variable region (LVR) that replicates efficiently in Plum Island porcine epithelial cells (PIPEC), a stable porcine cell line. Interestingly, the genomic changes associated with the replication of ASFV-G-ΔI177L/ΔLVR in PIPEC remained unaltered even after 30 serial passages in PIPEC. ASFV-G-ΔI177L/ΔLVR is equivalent to ASFV-G-ΔI177L with regard to safety, immunogenicity, and protective efficacy. ASFV-G-ΔI177L/ΔLVR is the first rationally designed, highly efficacious ASF vaccine candidate adapted to replicate in an established cell line, making it suitable for commercial vaccine production.
Adaptation of ASFV-G-ΔI177L to replicate in PIPEC.
ASFV-G-ΔI177L does not replicate in PIPEC, but virus yields are approximately 106 to 107 50% hemadsorption doses (HAD50)/ml in primary swine macrophages (Fig. 1A). The first passage of ASFV-G-ΔI177L in the PIPEC line was performed using a multiplicity of infection (MOI) of 10, and the infection rate was monitored by fluorescence microscopy detecting the expression of the mCherry cassette present in ASFV-G-ΔI177L. After 6 successive passages in PIPEC, a clear cytopathic effect began to occur. The virus was passed one additional time before stock virus was generated at passage 7, at which time ASFV-G-ΔI177LΔLVR reached a titer of approximately 106 HAD50/ml (Fig. 1B). Successive passages increased virus yields, with approximately 106 to 107 HAD50/ml achieved at passage 11 (Fig. 1C), confirming that the virus had adapted to replicate efficiently in the cell line.
Replication of ASFV-G-ΔI177L/ΔLVR in primary swine macrophages.
Swine macrophages are the primary cell target during ASFV infection in pigs (1). Previous attempts at adapting ASFV to replicate in a cell line have resulted in the concomitant inability to replicate in primary swine macrophages (12). The correlation between the ability of an attenuated ASFV strain to replicate in vivo and its ability to induce protection against disease has been documented in many studies (2–8, 12, 13). Therefore, it was critical to assess if ASFV-G-ΔI177L/ΔLVR can also replicate in swine macrophages. The in vitro replication characteristics of ASFV-G-ΔI177L/ΔLVR were evaluated in primary swine macrophage cultures and compared to those of parental ASFV-G-ΔI177L in a multistep growth curve. Two different passages of ASFV-G-ΔI177L/ΔLVR in PIPEC, the 7th and 11th passages (ASFV-G-ΔI177L/ΔLVRp7 and ASFV-G-ΔI177L/ΔLVRp11, respectively), were tested. Cell cultures were infected at an MOI of 0.01, and samples were collected at different times postinfection (Fig. 1B and C). The results demonstrated that both passages 7 and 11 of ASFV-G-ΔI177L/ΔLVR displayed growth kinetics similar to that of the parental virus ASFV-G-ΔI177L. Therefore, the adaptation of ASFV-G-ΔI177L to replicate in PIPEC, along with the acquired genomic deletion, does not significantly affect the ability of ASFV-G-ΔI177L/ΔLVR to replicate in vitro in primary swine macrophage cultures.
Genomic changes accompanying the adaptation of ASFV-G-ΔI177L to replicate in PIPEC.
The genomic changes acquired during the adaptation of ASFV-G-ΔI177L in PIPEC were assessed in the virus obtained after the7th passage (ASFV-G-ΔI177L/ΔLVRp7) using next-generation sequencing (NGS). Compared to the parental virus ASFV-G-ΔI177L, a deletion of 10,842 bp occurred between positions 16818 and 27660 of the ASFV-G-ΔI177L/ΔLVR genome. This genomic modification fully deletes the following genes belonging to the multigene family (MGF): MGF360-6L, MGF300-1L, MGF300-2R, MGF300-4L, MGF360-8L, MGF360-9L, and MGF360-10L. In addition, the genomic modification causes the deletion of the N-terminal portion of the MGF360-4L protein and the C terminus of the MGF360-11L protein. This deletion results in the creation of a novel hybrid protein, MGF360-4L/11L. The resulting ORF, which resides on the reverse coding strand, combines 830 nucleotides (nt) of MGF360-11L with 601 nt of MGF360-4L; thus, it is composed of 1,431 nt encoding a novel 476-amino-acid protein. One additional gene not belonging to the MGF, the X69R gene, is also deleted (Fig. 2). Altogether, eight genes are deleted, with a fusion of two additional genes.
The genomic stability of ASFV-G-ΔI177L/ΔLVR was further assessed in the population of virus obtained after passage 30. NGS analysis demonstrated no major additional genomic changes from the virus obtained after the 7th passage and, after continuation to 20 and 30 passages, determined that there was only one additional mutation in the E119L protein at position 167044, changing a serine to a threonine, and a nucleotide change in B438L that did not change the corresponding amino acid sequence. All other point mutations occurred outside of any ORF. This indicates that the genomic changes that occurred early in the process of adaptation allowed for efficient replication of ASFV-G-ΔI177L/ΔLVR in the PIPEC line.
Assessment of ASFV-G-ΔI177L/ΔLVR replication in swine.
To evaluate if changes in the ASFV-G-ΔI177L/ΔLVRp11 genome affected the attenuated phenotype of parental virus ASFV-G-ΔI177L, a group of five 80- to 90-lb pigs were inoculated intramuscularly (i.m.) with a high dose (106 HAD50) of ASFV-G-ΔI177L/ΔLVRp11 and were observed for 28 days. An additional, mock-inoculated animal was also included in the group to act as a sentinel, to test for the presence of virus shedding from the ASFV-G-ΔI177L/ΔLVR-inoculated animals. The five inoculated animals, as well as the sentinel, remained clinically normal and disease-free during the entire observation period, indicating that ASFV-G-ΔI177L/ΔLVR remains completely attenuated in vivo (Table 1 and Fig. 3).
TABLE 1 Swine survival and fever responses following infection with different doses of ASFV-G-ΔI177L/ΔLVR
Expt and dose (HAD50) of ASFV-G-ΔI177L/ΔLVR
No. of survivors/total no. of animals
Mean (SD) maximum daily temp (°F)
First expt, 106
The induction of protection by live attenuated viruses (LAVs) is usually linked to the ability of the virus to replicate after inoculation (2–8, 12). To assess the replication of ASFV-G-ΔI177L/ΔLVR in inoculated pigs, we quantified virus titers at different times postinfection (p.i.). Infected animals presented mild viremia (103 to 105.5 HAD50/ml) at day 4 p.i., reaching peak titers (103.5 to 107 HAD50/ml) by days 7 and 11 p.i., after which titers decreased (102.5 to 104 HAD50/ml) until day 28 p.i. (Fig. 4). Therefore, ASFV-G-ΔI177L/ΔLVR maintained the same complete attenuation phenotype as the parental virus ASFV-G-ΔI177L (6), with the infected animals presenting long viremias with relatively low values. In addition, all blood samples as well as spleens of the sentinel animals were negative by virus titration (sensitivity, ≥1.8 HAD50/ml), indicating the absence of virus shedding from the animals infected with ASFV-G-ΔI177L/ΔLVR.
Protective efficacy of ASFV-G-ΔI177L/ΔLVR against challenge with parental ASFV-G.
The ability of ASFV-G-ΔI177L/ΔLVR to protect animals against challenge with the virulent parental virus ASFV-G was tested at 28 days postinfection. Animals were challenged with 102 HAD50 of ASFV-G by the i.m. route. An additional group of five naive animals was challenged as a mock-inoculated control group.
As expected, all control animals displayed ASF-related signs beginning at day 4 postchallenge (4 dpc), with an increased severity in clinical signs until euthanasia by 7 dpc (Table 2 and Fig. 4). Conversely, animals infected with ASFV-G-ΔI177L/ΔLVR remained clinically normal with no signs of disease during the 21-day observational period. ASFV-G-ΔI177L/ΔLVR efficiently protected animals against disease when they were challenged with the highly virulent parental virus.
TABLE 2 Survival and fever responses of ASFV-G-ΔI177L/ΔLVR-infected animals challenged at 28 dpi with 102 HAD50 of virulent ASFV-G
Viremia values from animals infected with ASFV-G were as expected, with high titers (107.5 to 108.5 HAD50/ml) on day 4 p.i., increasing (averaging 108.5 HAD50/ml) by day 7 p.i., when all animals were euthanized. Conversely, viremia measurements after challenge in all ASFV-G-ΔI177L/ΔLVR-infected animals progressively decreased until the end of the experimental period (21 days after challenge), when, importantly, no circulating virus could be detected in blood from any animals (Fig. 4).
To investigate the effectiveness of ASFV-G-ΔI177L/ΔLVR at inducing protection against challenge, groups of five pigs were inoculated i.m. with decreasing doses of ASFV-G-ΔI177L/ΔLVRp11: 106, 104, and 102 HAD50 per animal. As a control, an additional group was inoculated with 102 HAD50 of ASFV-G-ΔI177L. In all cases, a noninoculated additional animal, a sentinel, was housed in the same room with the inoculated animals to assess the presence of virus shedding from the infected animals. As in the previous experiment, changes in body temperature and the potential presence of ASFV-related signs were recorded. None of the animals presented with clinical ASF disease during the 28-day observation period (Table 1 and Fig. 3).
Viremia kinetics in animals infected with 106 HAD50/ml of ASFV-G-ΔI177L/ΔLVR were nearly identical to those recorded in the previous experiment: mild viremia values by 4 days p.i. (dpi) (103 to 105 HAD50/ml), peaking (103 to 107 HAD50/ml) by days 7 and 11 p.i., and then decreasing (102.5 to 104 HAD50/ml) by day 28 p.i. (Fig. 4). Animals inoculated with 104 HAD50/ml of ASFV-G-ΔI177L/ΔLVR presented with viremia values that were slightly lower, though statistically similar to those of animals inoculated with 106 HAD50/ml of ASFV-G-ΔI177L/ΔLVR. Viremia values in animals inoculated with 102 HAD50/ml of ASFV-G-ΔI177L/ΔLVR were similar to those of animals receiving 104 HAD50/ml of ASFV-G-ΔI177L/ΔLVR, except that viremia was not detected until 7 dpi, and viremia titers at 11 dpi were significantly lower. Finally, animals inoculated with 102 HAD50/ml of ASFV-G-ΔI177L had titers with a kinetics profile very similar to that of animals receiving the same dose of ASFV-G-ΔI177L/ΔLVR.
In summary, ASFV-G-ΔI177L/ΔLVR-infected animals had low-to-moderate titers that persisted throughout the 28-day observational period. No virus was detected in any of the samples (blood samples at all time points as well as tonsil and spleen samples obtained at 28 days p.i.) obtained from sentinel animals (data not shown), indicating that ASFV-G-ΔI177L/ΔLVR-infected animals did not shed enough virus to infect naive pigs during the 28 days of cohabitation.
To assess the protective effects of the different doses of ASFV-G-ΔI177L/ΔLVR, all groups were challenged at 28 dpi with 102 HAD50 of ASFV-G by the i.m. route. Five naive animals were challenged as a mock-inoculated control group. All mock-inoculated animals started showing disease-related signs by 4 dpc, and disease severity evolved rapidly, with all animals euthanized by 7 dpc (Table 2). Conversely, all groups of animals infected with either ASFV-G-ΔI177L/ΔLVR or ASFV-G-ΔI177L remained disease-free and clinically healthy.
Viremia values in control animals infected with ASFV-G were, as expected, high (107 to 108 HAD50/ml) on day 4 p.i. and increased (averaging 108.5 HAD50/ml) by day 7 p.i., when all animals were euthanized. After challenge, viremias in ASFV-G-ΔI177L/ΔLVR- or ASFV-G-ΔI177L-infected animals progressively decreased until the end of the experimental period (21 days after challenge), when no circulating virus could be detected in blood from animals inoculated with either 106 HAD50/ml of ASFV-G-ΔI177L/ΔLVR or 102 HAD50/ml of ASFV-G-ΔI177L, and values remained very low (102.5 to 103.5 HAD50/ml) in animals inoculated with 102 or 104 HAD50/ml of ASFV-G-ΔI177L/ΔLVR (Fig. 4).
As in the previous experiment, no virus was detected in any of the blood samples or in tonsil and spleen samples obtained at 28 days p.i. from sentinel animals, indicating that no ASFV-G-ΔI177L/ΔLVR-infected animals shed enough virus to infect naive pigs during the 28 days of cohabitation.
In addition, at 28 days postchallenge, the presence of the challenge virus was detected, using a differential PCR that specifically identifies the ASFV-G strain (6), in two out of the five animals in the groups receiving 102 or 104 HAD50 of ASFV-G-ΔI177L/ΔLVR. Conversely, no challenge virus was detected in the spleens and tonsils of any of the five animals receiving 106 HAD50 of ASFV-G-ΔI177L/ΔLVR, indicating that high doses of ASFV-G-ΔI177L/ΔLVR can induce sterile immunity.
Therefore, ASFV-G-ΔI177L/ΔLVR induces protection even when administered in doses as low as 102 HAD50/ml. These results indicate that ASFV-G-ΔI177L/ΔLVR is as effective as ASFV-G-ΔI177L in inducing protection against challenge with the virulent parental virus ASFV-G.
Host antibody response in animals infected with ASFV-G-ΔI177L/ΔLVR.
Our previous experience working with different attenuated ASFV strains indicates that the only immunological parameter consistently associated with protection against challenge is the level of circulating antibodies induced by those strains (27). Therefore, we assessed the ability of ASFV-G-ΔI177L/ΔLVR to induce a circulating ASFV-specific antibody response and compared that with the response elicited in ASFV-G-ΔI177L-infected animals. An ASFV-specific antibody response was detected in the sera of these animals using two in-house-developed direct enzyme-linked immunosorbent assays (ELISAs). Animals infected with the higher doses (104 and 106 HAD50) of ASFV-G-ΔI177L/ΔLVR presented circulating antibodies as early as 7 days postinfection, reaching a solid plateau by day 11 p.i. with titers that were maintained until the day of the challenge. Animals receiving 102 HAD50 of either ASFV-G-ΔI177L/ΔLVR or ASFV-G-ΔI177L presented a delayed antibody response that was established by day 14 p.i. (Fig. 5). These results agree with previous reports of studies using low doses of ASFV-G-ΔI177L (6), supporting the observation that ASFV-G-ΔI177L/ΔLVR is able to induce an ASFV-specific immune response comparable to that induced by the parental virus ASFV-G-ΔI177L. It should be mentioned that no antibodies were detected in any serum sample obtained from any of the sentinel animals, confirming that sentinel animals were not infected by ASFV-G-ΔI177L-infected animals in any of the three groups.
The only efficacious experimental vaccine candidates for the current pandemic strain of ASFV are live attenuated strains developed by deleting specific virulence-associated viral genes (3–7, 11). The safety and efficacy of ASFV-G-ΔI177L make this experimental vaccine a leading candidate over other live attenuated strains for its potential to be commercialized.
A major limitation to considering ASFV-G-ΔI177L as a potential commercial candidate is the concern with regard to all LAVs for ASFV: the ability to replicate efficiently only in primary swine macrophage cultures. The adaptation of ASFV-G-ΔI177L to replicate in a continuous cell line without affecting its safety and efficacy as a vaccine candidate constitutes a major achievement and a major step toward its commercial production.
In this study, we report the ability of ASFV-G-ΔI177L/ΔLVR to replicate in PIPEC, a continuous cell line, abolishing the restriction of using only primary swine macrophages for the amplification of a candidate ASFV vaccine strain. ASFV-G-ΔI177L/ΔLVR maintained all the desirable ASFV-G-ΔI177L characteristics originally reported: it induces protection even when administered at low doses (102 HAD50), lacks any residual virulence for pigs even when inoculated at high doses (106 HAD50), does not shed to sentinel animals, and induces sterile immunity when used at optimal doses.
As in the case of ASFV-G-ΔI177L, all ASFV-G-ΔI177L/ΔLVR-infected animals still presented viremias by 28 dpi, in some cases with relatively high titers. The presence of long viremias or virus persistence is not a rare event in animals infected with attenuated ASFV strains. In our experience, the presence of viremia after inoculation is a characteristic of all attenuated virus strains effectively protecting animals against challenge with virulent isolates (2–10).
It appears that the genetic modifications responsible for the ability of ASFV-G-ΔI177L/ΔLVR to replicate in PIPEC do not significantly affect its replication in swine macrophages in vitro, in primary macrophage cultures, and, importantly, in vivo during infection in pigs.
Adaptation of ASFV to replicate in established cell lines is usually accompanied by attenuation of virulence and significant modifications to the viral genome. Different deletions in both variable regions of the ASFV genome have been described for viruses that have been adapted to replicate in cell lines, as well as in some naturally attenuated virus isolates.
Specifically, deletions encompassing regions within the left variable ends of the ASFV genome have been observed in the genomes of several cell culture-adapted viruses. For example, L60V (Vero cell-adapted L60) had three MGF110 genes deleted (14); Ba71V (Vero cell-adapted Badajoz 60) had genomic mutations throughout the genome (15); and Vero cell-adapted ASFV-G had deletions in multiple multigene family (MGF) genes (12). E70Ms14 (MS monkey kidney cell-adapted E70) and CV1 (E75 adapted to CV1 cells) viruses contained deletions in the terminal regions of the genome, although the exact genes are unknown (16).
In the case of known MGF deletions involved in cell culture adaptation, typically members of MGF505 and MGF360 are affected. Adaptation of ASFV-G-ΔI177L/ΔLVR resulted in deletion of MGF360 genes 4L, 6L, 8L, 9L, 10L, and 11L and MGF300 genes 1L, 2R, and 4L. Comparison of the MGF360 and MGF300 genes with those isolates that have been annotated (Fig. 6) revealed that the gene deletions in ASFV-G-ΔI177L/ΔLVR are unique. MGF300 genes 1L, 2R, and 4L, which are deleted in ASFV-G-ΔI177L/ΔLVR, are present in all annotated ASFV genomes, including the genomes of other tissue culture-adapted viruses. MGF360-8L is present in all sequenced isolates but deleted in ASFV-G-ΔI177L/ΔLVR. MGF360-4L is absent only from the ASFV Badajoz isolate adapted to Vero cells (BA71V). MGF360-6L is absent from the attenuated field isolate OURT88/3 and the Vero cell-adapted strain NHV BA7V. MGF360-9L, −10L, and −11L are absent or truncated in the attenuated recombinant Pr4Δ35 (17), Vero cell-adapted strain Ba71V (15), and attenuated field isolates OURT88/3 (18) and NH/P68 (19). In addition, the recently characterized protein X69R was also deleted in ASFV-G-ΔI177L/ΔLVR. We have reported that deletion of X69R has no effect on virus virulence (20).
The genomic changes observed in PIPEC-adapted ASFV-G-ΔI177L/ΔLVR constitute the first reported adaptation of an ASFV isolate or vaccine virus to cells of swine origin. It is possible that the differences between the genetic modifications observed in this study and those in other studies are due to the use of a cell line of nonswine origin. It is possible that some specific MGF genes are required for replication in swine cells, providing support for the observation that PIPEC, due to their swine origin, maintained the ability of ASFV-G-ΔI177L/ΔLVR to replicate in swine epithelial cells.
The results presented here demonstrate that ASFV-G-ΔI177L/ΔLVR, while able to replicate efficiently in an established cell line, retains all the characteristics of ASFV-G-ΔI177L in terms of safety, immunogenicity, and efficacy in protecting domestic pigs against ASF disease. To our knowledge, this is the first report of an experimental ASFV vaccine that has been adapted to replicate efficiently in a continuous cell line. Importantly, serial passages of ASFV-G-ΔI177L/ΔLVR did not alter the virus genome, supporting the possibility of large-scale production of ASFV-G-ΔI177L/ΔLVR as a vaccine strain.
MATERIALS AND METHODS
Cell culture and viruses.
Primary cultures of swine macrophages were prepared from swine blood by following procedures described previously. The preparation of macrophage cultures in 96-well plates for virus titration was also performed as described previously (21). PIPEC are a cell subclone derived after >60 passages from the LFPKαVβ6 cell line, a porcine fetal kidney cell line engineered to express bovine αVβ6 integrin (22). PIPEC cultures are maintained using Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Grand Island, NY) with 10% heat-inactivated fetal bovine serum (HI-FBS; Thermo Scientific, Waltham, MA) and Antimycotic (Life Technologies) at 37°C under 5% CO2.
ASFV-G-ΔI177L/ΔLVR was generated from serial passage of the live attenuated vaccine candidate strain ASFV-G-ΔI177L (6) as described in Results.
Comparative growth curves of ASFV-G-ΔI177L and ASFV-G-ΔI177L/ΔLVR were performed in primary swine macrophage and PIPEC cultures. Preformed monolayers were prepared in 24-well plates and were infected at an MOI of 0.01 (based on the HAD50 previously determined in primary swine macrophage cell cultures). After 1 h of adsorption at 37°C under 5% CO2, the inoculum was removed, and the cells were rinsed twice with phosphate-buffered saline (PBS). The monolayers were then rinsed 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 titers (expressed in HAD50 per milliliter) in primary swine macrophage cell cultures. All samples were run simultaneously to avoid interassay variability.
Virus titration was performed on primary swine macrophage cultures in 96-well plates. Virus dilutions and cultures were performed using macrophage medium. The presence of virus was assessed by hemadsorption (HA), and virus titers were calculated by the Reed and Muench method (23).
ASFV Georgia (ASFV-G), used in the animal challenge experiments, is a field isolate kindly provided by Nino Vepkhvadze of the Laboratory of the Ministry of Agriculture (LMA) in Tbilisi, Republic of Georgia.
Next-generation sequencing of ASFV genomes.
ASFV DNA was extracted from infected cells and quantified as described previously (24). The full-length sequence of the virus genome was determined as described previously using an Illumina NextSeq 500 system (24).
Animal experiments were performed under biosafety level 3 (BSL-3) conditions at the Plum Island Animal Disease Center (PIADC) facility, following a protocol approved by the Institutional Animal Care and Use Committee (IACUC) (protocol 225.01-16-R_090716).
Blood for the harvest of primary swine macrophages was collected under BSL-3 conditions in the animal facilities at PIADC. All experimental procedures were carried out in compliance with the Animal Welfare Act (AWA), the 2011 Guide for the Care and Use of Laboratory Animals (25), the 2002 PHS Policy for the Humane Care and Use of Laboratory Animals, and U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research and Training (IRAC 1985), as well as specific animal protocols reviewed and approved by the PIADC Institutional Animal Care and Use Committee of the U.S. Departments of Agriculture and Homeland Security (protocols 205.03-17-R, and 225.02-19-R, approved on 28 September 2017 and 10 September 2019, respectively).
The protective efficacy of ASFV-G-ΔI177L/ΔLVR was assessed using 80- to 90-lb commercial-breed swine. Groups of pigs (n = 5) were inoculated intramuscularly (i.m.) either with 102 to 106 HAD50 of ASFV-G-ΔI177L/ΔLVR or with 102 HAD50 of ASFV-G-ΔI177L. Clinical signs (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea, and cough) and changes in body temperature were recorded daily throughout the experiment. Animals inoculated with either ASFV-G-ΔI177L/ΔLVR or ASFV-G-ΔI177L were challenged i.m. 28 days later with 102 HAD50 of the virulent parental strain ASFV-G. The presence of clinical signs associated with the disease was recorded as described previously (6).
Detection of anti-ASFV antibodies.
ASFV antibody detection used an in-house indirect ELISA, developed as described previously (6). Briefly, ELISA antigen was prepared from ASFV-infected Vero cells. MaxiSorp ELISA plates (Nunc, St. Louis, MO, USA) were coated with 1 μg per well of an 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, St. Louis, MO). ASFV-specific antibodies in the swine sera were detected by an anti-swine IgM- or IgG-horseradish peroxidase conjugate (Kirkegaard & Perry Laboratories [KPL], Gaithersburg, MD, USA) and SureBlue Reserve peroxidase substrate (KPL). Plates were read as the optical density at 630 nm (OD630) in an ELx808 plate reader (BioTek, Shoreline, WA, USA). Titers in serum were expressed as the log10 of the highest dilution, where the OD630 reading of the tested sera at least duplicates the reading of the mock-infected sera.
The sequence of ASFV-GΔI177LΔLVR is available in GenBank under accession no. MW701371.
We thank the Plum Island Animal Disease Center Animal Care Unit staff for excellent technical assistance. LFPKαVβ6 (22) treated to remove bovine viral diarrhea virus (BVDV) was kindly provided by Mike LaRocco, Giselle Medina, and Teresa De Los Santos. We particularly thank Melanie V. Prarat for editing the manuscript.
This research was supported in part by an appointment to the Plum Island Animal Disease Center (PIADC) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract DE-SC0014664. This project was partially funded through an interagency agreement with the Science and Technology Directorate of the U.S. Department of Homeland Security under awards 70RSAT19KPM000138 and 70RSAT19KPM000056.
All opinions expressed in this paper are those of the authors and do not necessarily reflect the policies and views of the USDA, ARS, APHIS, DHS, DOE, or ORAU/ORISE.
Douglas P. Gladue and Manuel V. Borca have a patent application filed by the U.S. Department of Agriculture for ASFV-G-ΔI177L/ΔLVR and for ASFV-G-ΔI177L as live attenuated vaccines for African swine fever (26).
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