Insights and progress on epidemic characteristics, pathogenesis, and preventive measures of African swine fever virus: A review

ABSTRACT

The African swine fever virus (ASFV) is the only giant double-stranded DNA virus known to be transmitted by insect vectors. It can infect pigs and cause clinical signs such as high fever, bleeding, and splenomegaly, which has been classified as a reportable disease by the WOAH. In 2018, African swine fever (ASF) was introduced into China and rapidly spread to several countries in the Asia-Pacific region, with morbidity and mortality rates reaching 100 percent, resulting in significant economic losses to the global pig industry. Because ASFV has large genomes and a complex escape host mechanism, there are currently no safe and effective drugs or vaccines against it. Therefore, it is necessary to optimize vaccination procedures and find effective treatments by studying the epidemiology of ASFV to reduce economic losses. This article reviews research progress on pathogenesis, genome, proteome and transcriptome, pathogenic mechanisms, and comprehensive control measures of ASFV infection.

Introduction

African swine fever (ASF) is an acute, virulent, common infectious disease of domestic and wild pigs caused by the African swine fever virus (ASFV) [1]. African swine fever was first discovered in Kenya in 1921 and initially appeared in sub-Saharan countries [2,3]. The clinical features of African Swine Fever primarily include high body temperature, cyanosis of the skin, general organ bleeding throughout the body, and a mortality rate of up to 100% [4]. Once a pig is infected with African swine fever, its feces and secretions contain large amounts of the virus, posing a serious threat to healthy farm pigs [5]. However, the virus is not contagious to humans and is not a zoonosis. ASFV is the only representative of the African swine fever virus (Asfarviridae) [6–8]. ASFV is classified into 25 genotypes based on sequence differences at the 3’end of the B646L gene. Spinard et al. suggest that the current 25 genotypes based on the p72 sequence alone should be reduced to only 6 [9]. ASF is very harmful and spreads in various ways, posing a severe threat to the global swine industry. Prevention and control of ASF depend on elucidating its pathogenic mechanism and epidemiological variability. Due to the lack of safe and effective vaccines and antiviral drugs, culling is the most effective method to control the epidemic [10]. Therefore, an urgent need is to research effective vaccines and medicines to protect against the outbreak and stop its spread. Because of the current trend of research on ASFV, it is essential to carry out epidemiological investigations of ASFV strains and differential diagnosis and develop comprehensive prevention and control measures in line with the current epidemiological situation. This article reviews the latest research on ASFV pathogenetic, epidemiological characteristics, genome, proteome and transcriptome, pathogenic mechanisms, and comprehensive prevention and control.

Pathogenetic

ASFV structure and function

African swine fever virus (ASFV) is a complex macromolecular virus and the only member of the Asfarviridae family [6–8]. Due to its unique morphology and structure, ASFV is essential in virology. The ASFV particles exhibit a multilayer, icosahedral symmetry with a diameter of approximately 175-200 nm [6,11]. When examined under an electron microscope, ASFV shows a distinctly layered appearance and a highly organized arrangement. The viral genome contains over 150 open reading frames (ORFs), predicted to encode more than 180 proteins involved in viral DNA replication, gene transcription, and assembly [12,13].

The ASFV particle consists of five structures from the inside to the outside: nucleoid, capsid, inner envelope, core-shell, and outer envelope, and the virus-encoded proteins are distributed in different regions of the virus structure [11] (Figure 1, Table 1, Table 2). The nucleoid of ASFV, about 70-100 nm diameter, belongs to the genomic DNA of the virus particle, which is terminated by a covalently cross-linked hairpin loop containing the viral genome, DNA-binding proteins (p10 and pA104R) and transcription-associated proteins [4,14]. In addition, the ASFV genome encodes several non-structural proteins that play essential roles in the viral genome’s transcription, replication, and evasion of host immunity [12]. The outside of the nucleoid is the core shell, a protein layer about 30 nm thick. The core-shell proteins are hydrolyzed from two viral polyprotein precursors, pp220 and pp62, and the polyprotein pp220 plays a crucial role in nuclear assembly, including genome envelopment and condensation of the viral core proteins [15,16]. The inner vesicular membrane of ASFV is derived from the endoplasmic reticulum (ER) structure. This membrane includes the viral proteins p54, p17/pD117L, pH248R, and p12/pO61R, which are also reported in the outer capsid [17–19]. Studies have shown that the protein p54, encoded by the E183R gene, is a crucial factor in forming the viral inner membrane [17]. During the viral replication process, p17 and p54 primarily contribute to constructing the inner membrane layer, while p12, pE248R, and pE199L proteins mediate virus entry into host cells [20,21]. In addition, it was found that the p22 protein may also be an essential component of the viral bilayer inner membrane [20]. The outer layer of the inner capsid is composed of viral capsid proteins, with p72/pB646L accounting for the most significant proportion, followed by pM1249L, p17/pD117L, p49/pB438L, and pH240R [11]. p72 monomers interact through a double jelly-roll structure to form homotrimers, which constitute the pseudo-hexameric capsid (also known as the p72 capsid) [21]. The outer membrane is the outermost structure of ASFV, which is acquired from the host cell membrane during outgrowth and is absent from intracellular viral particles and viral particles released during cell lysis [14]. pE402R homolog is the only marker molecule for the external structure of the virus [6]. p12 acts as an outer envelope protein to facilitate the adsorption of viral particles on the host cell by binding to specific receptors on the host cell membrane and mediating the entry of ASFV [12]. The presence of an outer membrane not only protects the integrity of the internal structure of the virus particle but also complicates the mechanism of virus adsorption, invasion, and outgrowth [21]. The virus is infectious regardless of the presence of an outer membrane.

Figure 1.

Particle structure of African swine fever virus.

Table 1.

Major structural proteins encoded by African swine fever virus.

Protein Localization	Protein Name	ORFs	Main Function
Outer envelope	CD2v	EP402R	Involved in the regulation of various cellular functions, such as vesicular transport and signal transduction, in vivo diffusion
	p12	O61R	Involved in virus adsorption process
Capsid	pE120R	E120R	Involved in virus transmission and transportation
	pH240R	H240R	Involved in virus assembly
	p17	D117 L	Involved in the assembly of the viral capsid layer
	p49	B438L	Structural proteins essential for viral particle infectivity
	p72	B646L	a major viral capsid protein and involved in viral adsorption and invasion
Inner envelope	pEP84R	EP84R	Involved in virus assembly
	pE199L	E199L	Participates in viral invasion and enhances infectivity; induces cell death
	pE248R	E248R	Participates in viral invasion and enhances infectivity
	pH108R	H108R	Involved in virus adsorption process
	p12	O61R	Involved in virus adsorption process
	p17	D117L	The capsid is tightly anchored to the endosome membrane to maintain viral icosahedral structure and viability
	p22	KP117R	Involved in gene replication and regulation of invasion
	p30	CP204L	an important protein antigens and involvement in viral invasion
	p54	E183L	Interacts with dynamin light chain and is involved in viral invasion; induces apoptosis
Core shell	pp20	CP2475L	Polyprotein precursor that constitutes the viral core-shell, hydrolyzed to p5, p15,p35 proteins
	PP62	CP530R	Polyprotein precursor that constitutes the viral core-shell, hydrolyzed to p8,p15,p34 proteins
	Ps273R	S273R	hyprolyzed pp220 and pp62 proteins
Nucleoid	p10	K78R	Binds to viral DNA and participates in DHA replication
	pA104R	A104R	Binds to viral DNA and participates in DHA replication

Table 2.

Major nonstructural proteins encoded by African swine fever virus.

Protein Name	ORFS	Main Function
pD1133L	D1133L	Involved in gene transcription of ASFV
pQ706L	Q706L
PG1304L	G1304L
pB962L	B962L
pB263R	b263R
pc315R	C315R
pI243L	I243L
RP1	NP1450L	Involved in gene transcription and mRNA processing
RP2	D205R
RP3	EP1242L
RP5	C147L
RP6	H356L
RP7	D339L
pO174L	C475L	Involved in mRNA processing
pEP424R	EP424R
pD250R	D250R
p1215L	I215L
pC926R	C926R	Involved in the initiation of DNA replication and changing the pattern of DNA replication
pG1211R	G1211R	Involved in the initiation of DNA replication
pE301R	E301R
pF1055L	F1055L
pP1192R	P1192R	Involved in the initiation of DHA Replication and maintenance of normal DHA topology
pE165R	E165R	Provided dNTP and energy requried for viral DNA replocation
pK196R	K196R
pA240L	A240L
pF134L	F134L
pF778R	F778R
pE248R	E248R
pNP868R	NP868R	Substrate recognition and catalysis; involved in mRNA 5’ end-capping process
pB263R	B263R	Localized RNA polymerase and initiated gene transcripation
pC315R	C315R	Synergizes with RNA polymerase to form a transcription initiation complex
pA179L	A179L	Regulated programmed cell death
pA224L	A224L
pEPI153R	EP153R
L83L	L83L	Inhibition of TLR3-mediated intrinsic immune processes and interferon production
pDP71L	DP71L	Regulated protein synthesis processes in host cells; associated with viral virulence
pA238L	A238L	Regulation of host cell protein expression system and cytokine transcription
12L	MGF 360	Inhibition of IFN-β, NF-КB mRNA transcription and promoter activity
pA276R	MGF 360	Inhibition of phosphorylation of the interferon regulatory factor IRF3
pA528R	MGF 505	Inhibition of IFN expression and JAK-STAT pathway

Physical and chemical properties

ASFV demonstrates notable stability in external environments, with the capacity to survive for extended periods, up to several months, in a range of substrates, including soil, dried manure, frozen meat, blood, and unprocessed pork products [22–25]. ASFV exhibits enhanced resilience to physical factors, with high temperatures capable of inactivating the virus. Specifically, a 70-minute exposure at 56°C and a 20-minute exposure at 60°C is sufficient to inactivate the virus, underscoring the pivotal role of temperature in influencing the survival of ASFV [23]. Additionally, research has demonstrated that ASFV is inactivated within 1.5 days in a dry outdoor setting and 2.5 days in a dry indoor environment [26]. Furthermore, ASFV can be inactivated entirely after 30 minutes of exposure to sunlight. In the management of pig farms, the ventilation and dryness of pig houses should always be maintained, and the length of sunshine should be increased as much as possible. This is important for preventing and controlling African swine fever and improving the biosecurity level of pig farms. In addition, it was found that 3600μw/cm² UV irradiation intensity could completely inactivate ASFV within 3s [27]. Moreover, the nucleic acids of ASFV in cell cultures could be destroyed within 60s under the UV irradiation of 3600μw/cm² intensity [27]. It can be observed that the UVC intensity is sufficient to inactivate ASFV instantaneously. UVC is a physical disinfection method, and microorganisms in water can be effectively inactivated within a short period under irradiation of adequate intensity. The presence of sewage represents a significant risk to the biosecurity of agricultural premises. The inactivation of microorganisms in water represents an essential strategy for preventing the spread of ASFV [28].

Disinfection describes a crucial strategy for avoiding the emergence, transmission, and dissemination of infectious diseases in pig farms. ASFV exhibits stability within a pH range of 4 to 11. However, exposure to highly acidic and alkaline environments has been observed to reduce the infectivity of the virus [27]. It has been demonstrated that all disinfectants, except for the iodic acid mixture solution, are efficacious in killing ASFV and rendering it incapable of adsorbing erythrocytes [29,30]. Under the recommended concentration and specific action time, most disinfectants can completely inactivate ASFV. This is a guide for selecting the disinfectant type, dilution concentration, and time of action in production.

ASFV is more stable and survives longer in the presence of organic matter. Davies et al. observed that ASFV in feces remained infectious after 12 days, during which the maximum temperature was 21°C [5]. A similar pattern was observed in a urine study. However, elevated ambient temperatures markedly reduced the survival time of ASFV [5]. When stored at 4°C, ASFV in soil remained infectious for up to 112 days, with the gene still detectable on day 210 [28]. Disinfectants are greatly influenced by external conditions, such as the time of action, temperature, and the presence of organic matter, all of which affect the disinfection effect [31,32]. Studies have shown that the higher the organic matter content, the weaker the inactivation effect of disinfectants. The inactivation effect of disinfectants on ASFV increases with increasing temperature [32]. Based on this, to use disinfectants more scientifically and efficiently, it is recommended that when disinfecting areas on the surface of swine farms containing organic matter such as blood and feces, the concentration of disinfectants should be increased appropriately, and the duration of action should be prolonged to inactivate the virus completely.

The study of the physicochemical properties of ASFV has provided invaluable scientific insights that help develop effective prevention and control measures, thereby reducing the risk of virus transmission of African swine fever. However, the complexity of ASFV means that control efforts must adopt a multi-pronged strategy and continually adapt to the new challenges presented by virus mutations and environmental changes.

Characteristics of ASF prevalence

Overview of ASF prevalence abroad

In 1921, Montgomery et al. first described African swine fever in Kenya, with a mortality rate approaching 100 percent, and reported that the disease was transmitted by African warthogs that carried but did not develop the disease [2]. Since then, the disease has spread. In 1957, the first outbreak of ASF occurred in Europe. The ASF epidemic in Europe was divided into two phases: the first phase was from 1957 to 1995. ASFV was first detected in Portugal in 1957. The spread of ASFV to Portugal was due to the contamination of food scraps from African airlines or docked ships in ports used to feed pigs, consequently leading to the ASF outbreak in Portugal [33,34]. Rapid spread to surrounding Western European countries ensued, with Spain being the first to report cases of ASFV, followed by Italy, France, Malta, Belgium, and the Netherlands [35,36]. Strict epidemic control measures eradicated ASF from all European countries except Sardinia in 1995 [37–39]. The second phase occurred after 2007 when ASF was transferred from Africa via a cargo ship of contaminated slaughterhouse waste to Georgia, where the first case of African swine fever was reported in June of the same year [40]. The untimely diagnosis of ASF led to the spread of African swine fever within the country [41]. Subsequently, ASF continued to spread consistently across the Caucasus region of Eastern Europe and into southern Russia in 2007, spreading north and east. During this period, ASF occurred in many areas of Russia’s neighboring countries and the western part of the country. In January 2014, the EU reported the first ASF outbreak in wild boar in Lithuania, a Nordic country bordering Belarus. Since 2014, ASF outbreaks have been reported in several Nordic countries, including Poland [42,43]. In 2018, ASF outbreaks were reported in several Eastern European countries, including Hungary, Bulgaria, and Ukraine [44]. Belgium, located in Western Europe, also reported wild boar cases in 2018, with over 200 boars infected. Although the exact source of infection has not been identified, the movement of vehicles and items containing infectious viruses may be the primary transmission source [45].

Subsequently, between 2012 and 2014, ASF entered the EU borders and reached countries such as Ukraine, Latvia, and Poland. From January 2018 to March 2021, several severe ASF outbreaks occurred in Europe, Asia (Korea, China, Vietnam, Myanmar, Timor, Philippines, Laos, India, and Indonesia), and Africa (South et al., and Tanzania) [11,46]. The severity of these outbreaks underscores the need for comprehensive research and effective control measures, potentially impacting disease control strategies significantly.

Overview of the prevalence of ASF in China

In 2018, an ASF outbreak was reported for the first time in China, and in 2019, the virus was isolated and identified as a genotype II ASFV that was highly homologous to ASFV Georgia-07, which caused an acute infection with up to 100 percent of field pig deaths [47,48]. In 2020, the epidemiological strains were again monitored, and it was found that some low-virulence genotype II strains emerged in addition to high-virulence genotype II mutant strains. Whole genome sequencing of the low virulence mutant strains revealed a deletion of the EP402R gene encoding CD2v, which resulted in the strains lacking hematopoietic adsorption (HAD) capacity [49]. These low-virulence strains cause partial mortality in pigs at high infection doses and persistent, non-lethal, subacute, or chronic disease at low doses [49–51]. Low-virulence strains have long incubation periods and are efficiently transmissible, making early diagnosis more difficult. In 2021, a genotype I strain was isolated from pig farms in Shandong and Henan provinces in China, which had no erythrocyte adsorption capacity, showed low virulence to the pig body surface, and was capable of causing necrotic skin lesions and joint swelling [51]. A study reported in 2023 found that recombinant viruses of genotypes I and II were isolated from pig farms in China, with the genomes of the recombinant viruses being 43.5% homologous to genotype I and 56.5% homologous to genotype II [52]. The recombinant virus is highly lethal and transmissible and can completely circumvent the immunoprotection induced by live attenuated vaccination against genotype II viruses, as obtained from animal infection experiments [52]. The emergence of this recombinant virus will bring more problems and challenges to preventing and controlling African swine fever in China.

ASFV genome, proteome, and transcriptome

Genome

The ASFV genome has a complex structure, ranging from 170kb to 193kb in length depending on the isolate, and can encode 151–167 proteins [7,53]. Differences in genome length between isolates may be related to differences in the number of tandem inverted repeat sequences, and the main reason for the differences is associated with the presence or absence of members of the five Multigene Families (MGF) (MGF100,110,300,360, and 505/530) encoded by ASFV [54,55]. The genome of ASFV can be divided into the left variable region (LVR), the central conserved region (CCR), and the right variable region (RVR) due to the different distributions of GC content [56]. The central area is relatively conserved, with slight differences between strains, and the two variable regions have greater genetic diversity than the CCR, with the insertion and deletion of the MGF gene as the primary mode of mutation [57]. There is an apparent positive correlation between the genetic diversity of ASFV and the differences in the insertion and deletion of the MGF gene [58]. Sequence analysis of MGF genes with close ASFV gene type II relatives, particularly MGF360 and MGF505, has shown that there are about 8.5% sequence differences between the MGF360 genes and about 9.6% sequence differences between the MGF505 genes, suggesting that the MGF genes have experienced different selective pressures, resulting in their functional and regulatory genetic evolutionary diversity [59]. In addition, the gene DP71L has either long (184AA) or short (71AA) fragments, contributing to the ASFV genome’s variable length [60]. The study showed that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the development of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The mutational analysis of viral DNA shows that deletion of Pol X increases the mutation frequency in macrophages. Thus, the reparative DNA Polymerase Pol X of the ASFV maintains viral genome stability in vivo [61]. Genes such as MGF110-1 L, MGF505-10 R, and MGF 360-21 R are more complex and show genetic diversity, suggesting that ASFV may use multiple mutation mechanisms to acquire new phenotypic traits [62]. Genomics is one of the significant strategies for studying biodiversity, and an in-depth study of the genomics of ASFV could help to understand the genetic variation of ASFV better.

Proteome

ASFV particles have an icosahedral multilayered structure, contain a variety of polypeptides of unknown function, have a large genome and a high number of expression products, and interact with various host proteins during replication [63]. One study analyzed the protein composition of highly purified extracellular ASFV particles by mass spectrometry. It localized several detected proteins by immuno-electron microscopy. It found that proteomic analysis identified 68 viral proteins and that this fraction of encoded proteins accounted for 39% of the genome’s coding capacity [6]. Classified by function, most proteins involve viral gene transcription (19%) and morphological alterations (24%) [6]. The remaining proteins are associated with viral entry into the host (4%), evasion of host defenses (3%), and maintenance of genome integrity (6%), and 10% are known proteins, with a large number of proteins of unknown function still accounting for about 34% of ASFV [6]. In addition to the ASFV-encoded proteins, 21 host proteins were detected in the viral particles, including four enzymes (ENO1, GAPDH, TPI1, GGT1), two major cytoskeletal proteins (ACTU, TUBB), three small molecule GTPases (RAB5C, CDC42, RHOA), and a large number of membrane proteins (CD9, SLC2A1, ANXA1, ANXA2, ANXA4, ITGB1, ITGA3, ITGAV) [6,62]. The study analyzed the expression of ASFV proteins in three different sources of mammalian cell lines WSL-HP, HEK293, and Vero cells by mass spectrometry and found that the proteins P11.5, PK145R, and PI73R were expressed at high levels in all cell lines [64]. It was hypothesized that these proteins play an essential role during ASFV infection, and therefore, P11.5, PK145R, and PI73R proteins should be focused on in subsequent studies [64]. dUTPase (deoxyuridine triphosphatase) is an enzyme that plays a crucial role in DNA replication and repair [65]. It is primarily responsible for deoxyuridine triphosphate (dUTP) hydrolysis to produce deoxyuridine diphosphate (dUDP) [66]. This process is essential for maintaining the intracellular balance between dUTP and dTTP (deoxythymidine triphosphate), as dUTP can be incorrectly doped up by DNA polymerases into newly synthesized DNA strands, leading to DNA damage and mutation. dUTPase is present in a wide range of organisms, including bacteria, fungi, plants, and animals, and it plays a vital role in cell cycle regulation, DNA replication, and repair [65,66]. Since dUTPase is essential in maintaining DNA stability, it has been used as a drug design target for several pathogenic microorganisms [66]. ASFV encodes PE165R, a homotrimer, as a highly specific dUTPase, for which the development of small-molecule compounds is expected to achieve antiviral effects [67]. Therefore, a good understanding of the proteomics of ASFV is essential for vaccine and drug development.

Transcriptome

ASFV is like poxviruses in that it replicates in the host cytoplasm and has many similarities in its transcriptional mode [19,54]. ASFV has a “self-sufficient” transcriptional mode, in which the transcription factors required for the initiation of transcription, RNA polymerase (RNAP), Poly(A) polymerase, and viral capsids, are obtained directly from the virus particles, allowing it to better adapt to the host cell environment and enable rapid replication, which is favoring their transmission between pigs and even through tick-borne transmission [68]. CAGE-seq and RNA-seq have been used to analyze and quantify the transcript levels of ASFV genes during infection [69]. Cackett et al. performed CAGE-seq analysis on Vero cells infected with ASFV-BA71V and identified 149 differentially expressed genes (DEGs) [70]. Without inhibitors, these genes were categorized as early or late genes based on differential expression between 5 and 16 h post-infection: 65 genes were classified as early genes, 84 genes as late genes, and seven genes were not differentially expressed [70]. In contrast, RNA-seq analysis identified 47 early genes, 56 late genes, and 51 genes that were not significantly differentially expressed [70]. Differential gene function analysis revealed genes involved in genome replication and late transcription, as well as MGFs associated with evasion of the host immune response as early genes and structural proteins required for the formation of new viral particles, as well as early transcription factors packaged in viral particles as late genes [70]. One study investigated the differentially expressed genes in low pathogenicity ASFV isolates T88/3 (OURT) and high pathogenicity Georgia2007/1 (GRG) infected pigs using the RNA-seq technique [71]. The results showed that transcripts of ASFV genes were detected in the whole blood of GRG-infected pigs [71]. In contrast, transcripts of ASFV genes were not detected in the blood of OURT-infected pigs, which may be related to the pathogenicity of the virulent strains [71]. Although GRG and OURT differ in pathogenicity, there is a high degree of overlap with highly expressed genes in the host, and a small number of differentially expressed miRNAs have been detected from GRG- and OURT-infected pigs [70,71]. Transcriptomics provides a basis for understanding the underlying mechanisms of ASFV gene expression and its interactions with the host, and NGS technology has also been used to map the 5’ and 3’ ends of ASFV genome-wide transcripts, increasing the understanding that ASFV regulates its gene expression.

Pathogenic mechanisms and pathological characteristics of ASFV

Pathogenic mechanisms

ASFV replication characteristics

Viruses often utilize cellular endocytosis to overcome the physical barrier of the cell membrane to enter the cell to complete the viral infection process [12,15]. The types of cellular endocytosis exploited by viruses depend on activating specific cellular signaling pathways driven by virus-cell interactions [12]. Therefore, understanding viral invasion and the associated mechanisms of signaling pathways is essential for understanding viral pathogenesis. ASFV is tropic for macrophages, and monocyte-macrophages are the primary target cells for viral infection [72]. In addition, ASFV can also infect secondary target cells in vivo, including vascular endothelial cells, hepatocytes, and epithelial cells [73].

Viral infection of host cells is a complex and highly specialized biochemical process that can usually be divided into six main stages [54] (Figure 2). Phase I adsorption, the first step in viral infection, is binding the viral particle to a specific receptor on the host cell’s surface [54,74]. This step is critical for viral specificity, as viruses recognize and bind to different cell surface receptors. At this stage, ASFV binds to the host cell’s receptor, completing the adsorption of the virus [74]. Phase II invasion: Once a viral particle binds to a host cell, it enters the cell internally; the entry mode depends on the virus type [54,74]. ASFV is internalized within 30 minutes after the completion of adsorption or invades the cell by endocytosis/micropinocytosis [74]. It enters early endosomes between 1 and 30 minutes after infection and is translocated to late endosomes between 30 and 90 minutes [74]. The third stage is uncoating. After the virus enters the cell, it needs to release its genetic material from the protein coat, a process known as uncoating [75]. Decapitation can be achieved by enzymatic reactions, pH changes, or other mechanisms that allow the viral DNA or RNA to be exposed and ready for replication [75]. ASFV completes its decapitation and releases its genome in the late endosome, and the virus can only function if the genome has been removed from the protein coat [75,76]. The fourth stage is biosynthesis, in which the virus uses the metabolic pathways and machinery of the host cell to replicate its genetic material and synthesize viral proteins [76]. Gene expression begins after the genome is released from the protein coat and is divided into early, intermediate, and late gene expression [76]. Early viral genes start to be expressed 4-6 h after ASFV has infected the host cell, during which time they mainly encode ASFV replication-associated proteins. Replication of the ASFV genome begins 6-8 h after infection [74–76]. Genome replication primarily occurs in the cytoplasm, but transient replication in the nucleus also occurs at an early stage [7]. The fifth stage is assembly, in which the newly synthesized viral genetic material and proteins are assembled into new viral particles in the host cell [12,76]. After replication, the structural proteins that make up the ASFV particle and the intermediate and late genes begin to be expressed at 8-16 h [12]. The virus can then form a new virus particle in the nucleus early. Between 16 h and 24 h, the virus particles are assembled in the viral factory [12]. The final stage is release [12,76]. Once the viral particles are assembled, they must be released from the host cell to infect new cells [76]. Outgrowth release was performed 24 hours after ASFV infection [12].

Figure 2.

Replication cycle of ASFV.

Receptor-mediated endocytosis and the clathrin-mediated endocytosis pathway are essential for ASFV-entering macrophages [77]. Cholesterol in the cell membrane is critical for successful ASFV infection, and phosphoinositide 3-kinase (PI3K) activity and actin-dependent endocytosis are also required [73]. In addition, it has been shown that ASFV can also enter cells through exocytosis [73]. Macrocytosis is dependent on actin rearrangements and also involves the activation of cholesterol, sodium/proton exchanger (Na/H), EGFR, PKC, phosphorylated PI3K, Pak1 kinase, and the small Rho-GTPase Rac1 [73,78]. It has been reported that monoclonal antibodies to CD163 can block the infectivity of ASFV in porcine alveolar macrophages [79]. However, the role of CD163 in ASFV infection remains controversial, and the expression of CD163 in non-susceptible cells does not increase cellular susceptibility to ASFV [80]. It indicated that the role of CD163 in viral infection is not indispensable and that other pathways may exist to mediate viral invasion [81]. It also suggested the importance of further studies on the relationship between ASFV invasion and cellular receptors [82].

ASFV replication is mainly dependent on its gene expression. ASFV disrupts the nucleolus and nuclear membrane in infection and degrades lamin A/C [83]. The resulting membrane fragments are then recruited to the replication site, and the nuclear pore protein p62 is recruited to the periphery, likely to facilitate the viral genome replication [83]. In addition, the target cell of ASFV is the macrophage, but since deoxyuridine 5’-triphosphate nucleotide hydrolase (dUTPase) is not present in this cell, ASFV’s dUTPase is essential for its replication [84]. The dUTPase in ASFV has been found to have a dual function, firstly it catalyzes the hydrolysis of deoxyuridine triphosphate (dUTP) to deoxyuridine nucleotides (dUMP) and pyrophosphate, which prevents the incorporation of dUTP into the viral genome, and secondly, it assists in the participation of thymidine biosynthesis in the production of the deoxyguanosine triphosphate (dTTP) precursor [66,67]. Because of the high levels of dUTP or dUTP/dTTP present in macrophages, sufficient dUTPase is required to cope with DNA damage during viral replication [85,86].

Mechanisms of ASFV immune escape and host interactions

As a complex DNA virus characterized by a large genome, diverse protein composition, and intricate structure, the African Swine Fever Virus has developed a highly sophisticated immune evasion mechanism over its prolonged evolutionary history [4]. ASFV encodes more than 150 proteins, many of which are associated with the immune escape of the virus, and a variety of proteins interfere with the host’s immune system by modulating crucial processes such as the interferon response, apoptosis, inflammatory response, and autophagy, thereby aiding the virus in escaping immune surveillance, increasing within the host, and causing disease [6,13]. ASFV engages with the host through four principal mechanisms: it disrupts antigen processing and presentation, inhibits the production of interferons and the expression of pro-inflammatory factors, influences host protein expression and the cell cycle, and modulates autophagy and apoptosis in host cells [13].

ASFV affects antigen processing and presentation

The process of antigen presentation is divided into endogenous processing and presentation pathway (Major histocompatibility complex I (MHC-I) pathway), exogenous processing and presentation pathway (MHC-II pathway), and cross-antigen presentation pathway [87]. The critical cells/molecules that regulate the antigen presentation pathway are also part of the immune escape strategy of ASFV [87]. The target cells of ASFV are mainly macrophages, and the expression of MHC- I and MHC- II molecules on macrophages affects antigen presentation and the development of protective T-cell immune responses [87]. Sequence analysis of one study showed that the ASFV protein EP153R is homologous to host cell proteins such as CD94, CD69, and Ly94A and that it has the presence of a C-type animal lectin-like structural domain that has the function of binding MHC-like molecules [88]. Further studies revealed that EP153R inhibited the surface expression of MHC-class I antigens but did not affect the synthesis, glycosylation, and degradation of MHC-I [88]. ASFV infection down-regulated the expression of macrophage histone proteases, which impaired antigen digestion and processing, and the inhibition of SLA-DMA and SLA-DMB hindered the maturation of MHC-II molecules [68]. Thus, ASFV delays T-cell activation by blocking antigen delivery. In addition, the CD2v protein encoded by the EP402R gene of ASFV, which is an analog of the host T-cell surface adhesion receptor CD2, is expressed in the later stages of ASFV infection and functions in cell adhesion, enhancement of viral virulence, and modulation of the immune response [89]. When ASFV infects macrophages, it inhibits the proliferation of other cells and the reaction of non-infected lymphocytes to schizogenic, and this inhibitory effect is disrupted when the EP402R gene is absent [89]. Thus, CD2v is involved in intracellular transport of the virus and erythrocyte adsorption, inhibiting lymphocyte proliferation and regulating T cell-mediated cellular immune responses.

ASFV inhibits interferon production and pro-inflammatory factor expression

The central target cell of ASFV is the macrophage, which can initiate an immune response by recognizing PAMPs through a series of PRRs, such as TLRs, and activating the IFN signaling pathway to secrete cytokines/chemokines [90]. IFN is a class of cytokines that induces an antiviral state in infected and uninfected neighboring cells, and the antiviral mechanism in vertebrates is highly dependent on the action of IFN [90]. IFN is a crucial mediator of the host’s natural immune response to viral infection, inducing the expression of hundreds of ISGs that may have direct antiviral activity and regulating both innate and adaptive immunity by activating immature DCs to enhance NK cell function and promoting the survival and effector functions of T and B lymphocytes [91]. As IFN-mediated macrophage activation poses a severe threat to ASFV, ASFV must evolve antagonistic strategies to counteract this response, leading to the evolution of ASFV genes that regulate the IFN response.

The MGF360 and MGF505/530 genes encoded by ASFV inhibit interferon expression and are currently the primary target genes for constructing gene-deleted vaccines [92]. A276R, a member of MGF-360, can suppress the activation of Nuclear Factor Kappa B (NF-κB) and Interferon regulatory factor 3 (IRF3) [92]. However, its inhibition of IFN-β transcription/translation is unrelated to the NF-κB pathway. The MGF360-12 L inhibits the interaction between nuclear transport proteins and NF-κB, disrupting the nuclear translocation of NF-κB and significantly attenuating the production of IFN [93]. Furthermore, MGF505-7 R (A528R) targets the activation and nuclear translocation of p65 to suppress TLR8-mediated NF-κB activity, thereby reducing the signaling pathways of interferons and inflammatory cytokines while promoting viral pathogenicity through the inhibition of the JAK1 and JAK2 signaling pathways [94]. Additionally, MGF505-11 R negatively regulates the cGAS-STING signaling pathway by interacting with STING, ultimately suppressing the production of type I IFN [95]. Gene deletion assays showed that MGF360-505 R knockdown of ASFV reduced IL-1β transcription by inhibiting the NF-κB signaling pathway, ultimately reducing apoptosis and viral replication levels [96]. In addition, it was shown that a variety of encoded proteins of ASFV, including MGF360-15 R, MGF360-13 L, MGF360-9 L, MGF360-11 L, MGF360-14 L, E120R, I215L, I267L, A528R, E248R, EP402R, E184L, etc., were able to inhibit interferon production, hinder the interferon signaling effects and inhibit the expression of inflammatory factors [93,94,97–99].

ASFV affects host protein expression and cell cycle

ASFV encodes proteins associated with RNA transcription and modification, yet it relies on the host’s protein synthesis machinery [12]. ASFV inhibits host protein synthesis by recruiting translation-related factors, redistributing mitochondria to the “virus factory,” and degrading host mRNA [12,69]. The DP71L gene encodes a late-expressed protein that plays a crucial role in modulating the synthesis of host proteins. pDP71L can bind to p-eIF2α and facilitate its dephosphorylation, thereby preserving its biological activity and promoting the continued synthesis of viral proteins [100]. Similarly, I215L encodes a protein that functions analogously to E2 ubiquitin-conjugating enzymes, resulting in the ubiquitination of specific host proteins and playing a significant role in the transcription of late viral proteins and DNA replication [61]. ASFV mRNA exhibits structural similarities to host mRNA, incorporating a 5’cap and a 3’poly(A) tail, reliant on cap-mediated translation initiation [101]. D250R is an early protein, currently the only ASFV protein known to possess decapping enzyme activity, capable of specifically binding to ribosomal protein L23a [101]. During the late stages of ASFV infection, D250R predominantly localizes to the “viral factory,” selectively targeting host mRNA and inhibiting the synthesis of both viral and host proteins. It is hypothesized to facilitate RNA release, thereby preventing the accumulation of dsRNA and excessive synthesis of viral proteins, which could trigger immune responses [69,101].

ASFV regulates host cell autophagy and apoptosis

Autophagy and apoptosis are stress responses that cells exhibit when subjected to external stimuli to eliminate aberrant cells and counter infections [74]. Premature apoptosis is detrimental to the proliferation of the African Swine Fever Virus, which is why multiple genes within ASFV can inhibit host cell apoptosis during the early stages of infection [74,102]. In cells infected with ASFV, indicators of apoptosis in host cells include DNA fragmentation, activation of caspase-3, and the release of cytochrome C from the mitochondria [74]. It has been demonstrated that ASFV infection induces caspase-9 activation via endoplasmic reticulum stress, which correlates with mitochondrial apoptotic signaling pathways and caspase-12 activity: endoplasmic reticulum stress causes a dramatic increase in intracellular Ca²⁺ concentration, leading to activation of the endoplasmic reticulum chaperones calreticulin and calreticulin, and increases the permeability of the outer mitochondrial membrane, resulting in CytC release [103]. ASFV infection of Vero cells also induces cleavage of DNA repair enzyme (poly ADP-ribose polymerase, PARP), a nuclear protein cleaved explicitly by caspase-3 and caspase-7, suggesting that this is the next activation pathway for ASFV-induced apoptosis [13]. ASFV pA224 L is a member of the inhibitor of apoptosis protein (IAP) family [104]. Studies have shown that pA224 L inhibits TNF-α-induced caspase-3 activation and apoptosis [105]. pA224 L overexpression activates the NF-κB pathway, which inhibits apoptosis and promotes viral proliferation by activating the transcription of many anti-apoptotic genes, including IAP and Bcl-2 family members [106]. ASFV pEP153R resembles the N-terminal structural domain of some C-type lectin molecules. pEP153R is a multifunctional protein that inhibits the expression of MHC-I molecules and prevents apoptosis or viral infection induced by astrocytes by blocking p53 protein activation [107]. Infection with the ASFV BA71V strain lacking the pEP153R gene (ASFV-ΔEP153R) activates caspase-3, which causes cell death. Interestingly, ASFV EP153R-infected cells lost the ability to adsorb erythrocytes, suggesting that pEP153R is also involved in erythrocyte adsorption [108].

Autophagy is a vital pathogen clearance mechanism other than apoptosis that directly degrades pathogens and makes their products available to the host cell [72]. ASFV-encoded A179L is a Bcl-2 homolog that interacts with pro-apoptotic Bcl-2 family proteins to inhibit not only apoptosis but also a homolog of ICP34.5 that inhibits autophagy by interacting with Beclin-1 through its BH3 structural domain [109]. In addition, Suresh et al. resolved the crystal structure of the A179L-bound Beclin-1 BH3 motif. They found that A179L binds to Beclin-1 and Bcl-2 through the same ligand-binding groove, that the K115 of Beclin-1 interacts with the D80 and E76 ion channels of A179L, and that mutation of both amino acids may reduce the binding of A179L to Beclin-1 binding [110]. The targeted mutant ligand-binding groove inhibits Beclin-1 binding to A179L, resulting in a loss of A179L’s ability to inhibit autophagic vesicle formation [110]. In addition, the ASFV-encoded protein E199L can induce an intact autophagic process in Vero and HEK-293T cells by interacting with a host reductase (pyrroline-5-carboxylate reductases, PYCR) [111].

Pathological characteristics

In infected pigs, the virus replicates in monocytes and macrophages, leading to swelling and rounding of cells and marginalization of nuclear chromatin [112,113]. Upon maturation, the virus induces cell death and rupture, releasing large numbers of viral particles into the bloodstream, lymph, and interstitial tissues [112,113]. After infection in the oral-nasal cavity or muscles, the virus replicates mainly in mononuclear phagocytes of the tonsils and lymph nodes, such as the jaws [114]. It spreads through the lymphatic and hematological circuits to replicate in target cells in other organs, including hepatocytes, capillary endothelial cells (kidney, liver), tonsillar epithelial cells, fibroblasts, smooth muscle cells, pericytes, mesenchymal stromal cells in the liver, glomerulonephritis, neutrophils, lymphocytes [114,115]. The virus replication and subsequent cell death can be observed in macrophages of various organs, resulting in a significant presence of free viral particles within the interstitial space [115].

Erythrocyte adhesion is a significant characteristic of the African swine fever virus [116]. During in vitro culture, erythrocytes are observed to adhere to the periphery of infected cells, and a similar phenomenon has been documented in vivo, where erythrocytes serve as a critical vehicle for the widespread dissemination of the virus throughout the body [116,117]. Furthermore, viral particles have been detected in lymphocytes and platelets, albeit without replication, suggesting that these cells may function as transport carriers akin to erythrocytes [117]. Additionally, elevated serum cytokines TNF-α and IL-1β coincided with the timing of the organism’s fever after ASFV infection [118,119]. Characteristic changes in acute ASF include petechial hemorrhages, varying degrees of bleeding in the gastrointestinal tract, liver and renal lymph nodes, splenomegaly, pulmonary edema, and disseminated intravascular coagulation[112,120]. Oedema and bleeding are more intense in subacute ASF than in acute ASF [112]. Ascites, pericardial effusions, and renal and gallbladder edema are also more common, and splenomegaly is frequently observed [120]. In subacute ASF, hemorrhage (petechiae and ecchymoses) is more frequent in the kidneys’ cortex, medulla, and renal pelvis than in acute [119,120]. Hemorrhages may also be observed in the stomach, liver, kidneys, and submandibular, retropharyngeal, mediastinal, mesenteric, and inguinal lymph nodes [120]. Pulmonary edema is the leading cause of respiratory distress, and the animal usually dies in shock; large amounts of foam can be observed in the mouth and nose[112,120].

Mode of transmission of ASFV

Direct transmission

Direct contact between infected and healthy pigs is the most common mode of ASFV transmission. Experimental studies have shown that direct contact with infected domestic pigs is an effective mechanism for ASFV transmission. ASFV-infected pigs and susceptible pigs were infected within 1 to 9d [121]. When healthy pigs were housed apart from infected pigs with solid partitions, susceptible pigs were infected within 6 to 15d [121]. Highly pathogenic ASFV strains prevalent in Lithuania, Georgia, and Russia infecting pigs can be detected in blood up to 109 HAD₅₀/mL (50% hematocrit) and in saliva, urine, or feces up to 105 HAD₅₀/mL [122]. Therefore, the virus can be excreted through saliva, nasal secretions, and feces, inhaled or ingested through the mouth and nose to infect new hosts [122].

Indirect transmission

The indirect transmission routes of ASFV primarily involve two significant pathways. Firstly, feeding waste containing infectious meat products [123]. Secondly, procuring infected pigs, contaminated bedding or feces, and swill through illegal trade channels [124]. In severe environmental contamination, polluted vehicles, equipment, and clothing can also facilitate the spread of ASFV [125]. Additionally, during vaccination and pharmacological treatment, inadequate disinfection practices or failure to replace contaminated needles may contribute to the transmission of ASFV [126]. The Food and Agriculture Organization of the United Nations (FAO) reported that the May 2007 ASF outbreak in Georgia was most likely caused by mishandling waste discarded from international ships carrying contaminated meat or meat products [123]. Sequence evolutionary analyses showed that the Georgian virus isolate was closely related to Mozambique, Zambia, and Madagascar [123]. In May 2008, a Russian pig farm was infected with ASFV, which spread to Leningrad, 1,000 km away, by road transport. ASF in Malta and Sardinia, Italy, was initially caused by feeding local pigs swill from an infected area [41].

Aerosol propagation

ASFV-infected pigs transmit the virus to the environment via excretions and secretions, with exceptionally high viral loads in oral fluid, nasal fluid, feces, and urine during the acute phase [126,127]. ASFV-positive pigs carrying high viral titers during sneezing and coughing can aerosolize and discharge their secretions into the environment [128]. Wilkinson et al. demonstrated that ASFV can be transmitted through aerosols from ASFV African strain-positive pigs, with infections occurring at distances of up to 2.3 m, providing preliminary evidence for airborne transmission of ASFV under experimental conditions [129]. The half-life of ASFV in aerosol under experimental conditions was approximately 14 minutes, and viral titers as high as 10^3.2 TCID₅₀/m³ were found in aerosol samples collected from the rooms of ASFV-positive pigs [129]. In addition, Li et al. found that the Asian strain of ASFV could be transmitted up to 10 m under field conditions [130].

Insect transmission

ASFV can replicate in ticks (Ornithodoros sp.), the most common carriers of the virus. These ticks live in the nests of wild boars, and adults can survive for decades without food, making the blunt-edged tick an ideal host for the virus. Studies have shown that the Georgia strain of ASFV can be replicated in wandering blunt-edged ticks, joint in southern Europe, and conserved for at least 12 weeks [131]. However, not all ticks are capable of transmitting ASFV, and studies have found that ASFV is unable to replicate in hard ticks (Ixodes ricinus and Ixodes reticulatus ticks), which are also common in Europe, suggesting that this family of ticks has limited vectorial capacity [132]. In addition, other blood-sucking insects, such as mosquitoes and flies, can mechanically transmit ASFV in infected areas [132,133]. Stable flies can transmit ASFV after 24 h of feeding on the blood of infected pigs and can carry high blood titers of ASFV for more than 48 h [133]. Sting flies of the genus Cnidaria have been shown in experiments to transmit ASFV to domestic pigs for a limited period mechanically [132]. ASFV has also been detected in pig blood lice, which are endemic in temperate regions [133].

Potential transmission by boar semen

An essential route of widespread transmission of animal diseases is through boar semen used for artificial insemination. Virginia et al. reported that ASFV can be efficiently transferred from infected boars to naïve recipient sows by artificial insemination (AI) [134]. The study showed that after extended semen insemination, 7 out of 14 reserve sows were positive for ASFV at seven days post-insemination, and all reserve sows were positive for ASFV at 35 days post-insemination [134]. Twelve out of 13 gestating reserve sows aborted or absorbed at the time of heat. A proportion of the remaining gilts showed abnormalities and replication of ASFV in fetal tissue [134]. The results emphasize the critical role of porcine semen in ASFV transmission. In modern pig production, semen from boars frequently supplies many sow herds. Thus, infection of boars poses the risk of rapid and widespread spread of ASFV within or between countries.

Intercellular transmission of ASFV using apoptotic vesicles

Intercellular transmission is an essential strategy for viruses to evade humoral immunity, and extracellular vesicles (EVs) have attracted much attention as crucial carriers of intercellular material exchange and information transfer. In 2023, Yang’s team investigated the infection and transmission of ASFV, which revealed that ASFV hijacks the apoptotic pathway of the host cell and uses apoptotic vesicles to carry out infection and intercellular transmission by new mechanisms [135]. Apoptotic vesicles are viral particles without an outer vesicle membrane, which can effectively escape the neutralizing effect of antibodies and are an essential mode of infection of ASFV. The results of this study provide a scientific basis for elucidating the escape mechanism of humoral immunity of ASFV and shed light on the creation of a new type of vaccine [135].

ASFV comprehensive control measures

Progress of vaccine research

Inactivated vaccine

Vaccine research against ASFV dates back to the 1960s when the first attempts to use inactivated vaccines against ASFV began due to the simplicity and safety of inactivated vaccine preparation. CadenasFernández et al. immunized pigs against a wide range of inactivated ASF antigens by traditional methods and found that, although they were able to induce a serological immune response in some cases, they ultimately failed to produce adequate protection [136]. Researchers at the International Livestock Research Institute (ILRI) in Kenya, in collaboration with Colorado State University (CSU), are now attempting to develop an inactivated vaccine using a new method of the Mirasol process, in which samples of the ASF virus are mixed with riboflavin (Vitamin B2) and then treated with a specific amount of ultraviolet light that breaks down nucleic acids in the virus, resulting in the loss of the virus’s ability to replicate, which is expected to lead to the preparation of ASF vaccines. Vaccine, but subsequent progress remains to be investigated.

Live attenuated vaccines

Live-attenuated vaccines (LAV) are the fastest way to develop safe and effective vaccines against African swine fever. Three main strategies have been used to produce the live-attenuated vaccine ASF-LAV: attenuation by cell passaging, screening for naturally attenuated strains, and deletion of virulence-related genes [137].

Successive passages of genotype I virulent ASFV strains in the porcine bone marrow and renal cells resulted in attenuation of cell passages, and pigs immunized with the attenuated strains were protected against the virulent strain yet subsequently developed chronic ASF. Balysheva et al. attenuated genotype II strains by passaging them in the porcine lymphoblastoid hybrid cell line, A4C2/9k, and in the African green monkey renal cell line, CV-1. Stavropol 01/08 strain, and although the resulting viruses lost pathogenicity, they failed to protect pigs from virulent attack [138].

Most pathogenic ASFV field isolates induce erythrocyte adsorption, a phenomenon in which erythrocytes adhere to the surface of virus-infected cells. However, several naturally low virulent ASFV isolates do not induce haemadsorption [139,140]. This observation led to the hypothesis that the viral genes mediating erythrocyte adsorption are the major virulence factors, and two genes, EP402 and EP153R, have been identified as responsible for the induction of erythrocyte adsorption [140]. It was found that ASFV mutants lacking CD2v exhibited a wide range of virulence phenotypes; deletion of CD2v alone in the genomes of Malawi Lil-20/1, ASFV-G, and HLJ/18 isolates did not significantly alter virus virulence. Monteagudo et al. found that the virulence of the deletion of the CD2v (EP402R) gene of the ASFV BA71 strain was highly attenuated in vivo, that vaccination of pigs with the deletion mutant virus BA71 ∆CD2v provided protection not only against lethal attacks of the parental BA71 but also against heterologous E75 strains, and that BA71 ∆CD2v-induced protection was dose-dependent, and that the in vivo the observed cross-protection correlated with the ability of BA71 ΔCD2v to induce specific CD8+ T cells capable of recognizing both BA71 and E75 viruses in vivo. Interestingly, BA71 ΔCD2v-immunized pigs were 100% resistant to lethal attack by Georgia 2007/1 [50]. The deletion of CD2v from the genome of ASFV-Kenya-IX-1033 partially reduced the virulence of the virus, and 100% of pigs survived vaccination but exhibited fever and decreased appetite. In addition, the deletion of CD2v from the genome of the ASFV strain BA71, which is adapted to grow in COS-1 cells, completely attenuated viral virulence[57,138].

Deleting virulence-related genes or evasion of the immune response by homologous recombination or CRISPR/Cas9 gene editing to improve the safety of naturally attenuated strains and the attenuation of circulating muscular virulent strains is essential for current ASF-LAV research [141]. The number of MGF genes varies widely between different ASFV strains. Naturally, low-virulence or cell culture-adapted ASFV strains tend to have fewer MGF genes than high-virulence strains, suggesting that these genes may not be essential for viral replication [142,143]. Previous studies have shown that MGF genes may determine ASFV tropism [144,145]. In addition, several MGF genes have been shown to suppress host innate immunity [146]. Therefore, MGFs are potential targets for the rational design of LAV vaccines. O’Donnell et al. constructed an ASFV G-ΔMGF deletion toxin by deleting six genes, MGF 360-12 L, MGF 360 - 13 L, MGF 360 - 14 L, MGF 505-1 R, MGF 505 - 2 R, and MGF 505 -3 R, using Georgia 2007/1 as the parental strain. The replication efficiency of ASFV-G-ΔMGF in culture in porcine primary macrophages did not differ from that of the parental strain, but it was utterly attenuated in pigs [147]. Intramuscular injection of pigs with a single dose of ASFV-GΔMGF provided complete protection against the lethal challenge of the highly virulent parental virus, with only a few pigs experiencing transient fever [147]. In addition, this vaccine candidate was tested in oral vaccination trials in feral pigs. The vaccine induced an immune response in 50% (4/8) of the vaccinated boars, and all responders were wholly protected from subsequent attack infections[140,150]. Seven genes, MGF505-1 R, MGF505-2 R, MGF505-3 R, MGF360-12 L, MGF360-13 L, MGF360- 14 L, and CD2v (HLJ/18-7GD), were deleted from the parental strain, ASFV HLJ/18, after a safety profile of pigs, commercial pigs, and pregnant sows free of specific pathogens, immunogenicity and protective effect evaluations showed that the deletion strains were completely attenuated in pigs and did not regain virulence, and provided complete protection against highly pathogenic ASFV attacks, which is expected to play a role in controlling the spread of ASF [49].

In addition to large deletions of multiple MGF genes, studies have attempted to assess the contribution of individual MGF genes to virus virulence and protection. The MGF 505-7 R gene inhibits host type I interferon induction through multiple mechanisms [90,151–152]. Deletion of MGF 505-7 R from a potent ASFV strain had differential effects on virus replication in primary porcine macrophage cultures and virus virulence in pigs [148,149]. Following intramuscular injection with low doses (10 HAD₅₀) of the ASFV-Δ MGF 505-7 R mutant, 100% of pigs survived, whereas 43% (3/7) of pigs inoculated with a high dose (10⁵ HAD₅₀) of the mutant died of infection [148,149]. Li et al. obtained a double deletion strain (ASFV-ΔH240R-Δ7 R) by deleting the MGF505-7 R and H240R genes using ASFV HLJ/18 as the parental strain. It was found that piglets immunized with ASFV-ΔH240R-Δ7 R were safe without any ASF-associated symptoms and produced specific antibodies against p30. Under challenge with the highly virulent ASFV HLJ/18, the immunized piglets in the high-dose group (10⁵ HAD₅₀) showed 100% protection without clinical signs, and no pathogenicity was observed by necropsy and histological analysis [150]. MGF 360-9 L inhibits IFN-β signaling by targeting STAT 1 and STAT 2 for degradation [151]. Deletion of MGF 360-9 L alone had no significant effect on ASFV replication in porcine macrophage cultures. Double deletion of MGF 360-9 L and MGF 505-7 R from the highly virulent ASFV strain CN/GS/2018 (genotype II) slightly reduced viral replication in porcine macrophages while attenuating viral virulence in pigs [152]. The resulting recombinant strain (designated ASFV-Δ9L7R) provided 83.3% (5/6 pigs) protection against lethal challenge with the parent virulent virus [152]. Numerous studies have demonstrated the effect of deleting MGF genes on ASFV virulence and protection, and the results may vary when the same set of MGF genes are deleted from different ASFV strains [140].

Early studies identified three highly conserved genes required for virulence: NL (DP71L), UK (DP96R), and 9GL (B119L) [153,154]. Deleting the NL gene from the potent ASFV strain E70 significantly reduced its virulence, but had no effect in the Malawi strain [154,155]. Deletion of NL in the Georgia 2010 (ASFV-G) strain, although replicating in porcine macrophages, showed severe replication defects in pigs and failed to induce protection against parental ASFV-G infection [155]. The UK (DP96R) gene encodes a 15kDa protein expressed early in the virus-infected cell. Deletion of DP96R from ASFV strain E70 or ASFV-G did not affect virus replication in primary porcine macrophage cultures, deletion of the UK gene from ASFV strain E70 significantly reduced its virulence, but deletion of the UK gene from ASFV-G did not reduce the virulence of the virus; therefore, the virulence of the virus by the UK gene depends on the strain [140,156,158]. The 9GL (B119L) gene is a homolog of ERV1 and plays a role in oxidative phosphorylation [156]. It was found that deletion of the 9GL gene from the genome of the ASFV Malawi Lil-20/1 strain severely affected virus replication in porcine macrophages. It resulted in a complete attenuation of virus virulence, and pigs immunized with this gene deletion mutant were protected against lethal infection by the parental strain [156].

The natural weak strain OURT88/3 has been shown to induce immunoprotecting. However, adverse reactions, including fever and joint swelling, have been observed in some pigs after vaccination [157]. Double deletion of these two genes does not significantly affect virus replication in macrophages in vitro but substantially reduces its protective potential [158]. Simultaneous deletion of the 9GL (B119L) and UK genes in the ASFV-G strain completely attenuated the virus. All pigs vaccinated with the double deletion mutant ASFV-G-Δ9GL/ΔUK survived and showed no significant clinical signs of ASF, even at a high dose of 10⁶ HAD₅₀. Vaccination with the ASFV-G-Δ9GL/ΔUK mutant provided complete protection against a lethal challenge with the parental virus, and protective immunity was achieved two weeks after vaccination [159]. However, when the NL gene was deleted from ASFV-G-Δ9GL/ΔUK, the resulting triple deletion mutant ASFV-G-Δ9GL/ΔNL/ΔUK appeared to be overly attenuated and failed to produce protective immunity within two weeks of vaccination [155]. Deletion of both the 9GL and UK genes in the ASFV HLJ/18 strain showed that similar to ASFV-G, the double deletion of 9GL and UK in ASFV HLJ/18 also resulted in complete attenuation of the virus in pigs[8,140]; in contrast to ASFV-G, pigs vaccinated against the double deletion mutant HLJ/18-Δ9GL/UK was not protected against parental virulence viruses during a lethal challenge[49,140]. This indicates that the 9GL and UK genes are the major virulence determinants. Simultaneous deletion of these two genes in genotype II ASFV strains (ASFV-G or HLJ/18) completely attenuates the virus. However, the protective efficacy of the resulting double deletion mutants was inconsistent [140].

Genetically engineered vaccines

ASF genetically engineered vaccines, including subunit, DNA, and live-vector vaccines, represent a beacon of hope in the fight against ASF. They offer a potential reduction in side effects and increased safety, paving the way for a more effective prevention strategy [23].

Subunit vaccines use purified recombinant proteins or synthetic peptides that contain specific viral epitopes capable of inducing a protective immune response. Gomez-Puertas et al. found that antibodies induced by the p72, p54, and p30 proteins had a neutralizing effect, with antibodies to p72 and p54 inhibiting viral adsorption and antibodies to p30 inhibiting viral internalization [160]. The first subunit vaccine shown to be protective against ASFV infection was the baculovirus-expressed ASFV CD2v protein. Neilan et al. used baculoviruses to express the p54, p72, p30, and p22 proteins of the highly virulent strain Pr4 and immunological pigs, which detected specific antibody production [161]. The Pr4 strain was then used for a takedown, and the immunized group showed only delayed onset of disease, with the recombinant proteins not providing adequate immune protection [161]. It has been found that antibodies induced by recombinant viral proteins play only a complementary role in immunoprotection and do not provide safe and effective immunoprotection for immutable animals [162]. In addition to antibodies, vaccines capable of stimulating T cell-mediated cellular immune responses may be needed to protect ASFV [162].

In contrast to antigen-based subunit vaccines, DNA vaccines play an important role in preventing ASFV by inducing cell-mediated CTL immune responses [163]. p54 is one of the major envelope proteins of ASFV and is involved in the adsorption and internalization process of the virus [163]. Studies have shown that DNA vaccines containing the p54 gene can induce specific antibodies and T-cell responses. However, DNA vaccines with the p54 gene alone have limitations regarding protective efficacy and usually do not fully protect pigs against highly virulent ASFV. Nevertheless, the p54 gene remains an essential target in ASFV DNA vaccine research. Similarly, DNA vaccines with the p30 and CD2v genes can induce some protection at high doses but are still insufficient to completely protect pigs against highly virulent ASFV [163].

The NA vaccine candidate pCMV-sHAPQ, a DNA vaccine candidate (sHA) encoding ASFV p30 and p54 fused to the extracellular structural domain of hemagglutinin (sHA), improves humoral and cellular responses in pigs but provides partial protection against lethal challenge with virulent E75 ASFV strains [164]. Similarly, immunization of pigs with plasmid constructs encoding p30, p54, and the ubiquitin-fused sHA gene elicited a T-cell response. Still, it partially protected against lethal challenges with virulent E75 virus strain without neutralizing antibodies. In this study, the protective effect correlated with the presence of sHA-specific CD8+ T cells [164,165]. In another experiment, immunization of pigs with a DNA expression library of more than 4,000 plasmid clones, each containing a random Sau IIIa restriction fragment extracted from viral genomic DNA and fused to ubiquitin, resulted in 60% protection against lethal challenge with virulent E75 [166]. It was also shown that immunization of pigs with recombinant proteins (p15, p32, p54, and ±p17) and plasmid DNA constructs encoding (p32, p72, EP402R, and ±p17] at primary and two booster doses induced a cell-mediated immune response and antibodies that were shown to neutralize ASFV in vitro [167]. However, the immunized pigs were not resistant to the Armenia 2007 strain.

Viral vector vaccines use safe and non-pathogenic virus vectors to express viral immunogenic proteins. These can induce strong specific antibody-mediated humoral immunity and IFN-γ-secreting cellular immunity in swine, partially adequately protecting against ASFV infection. One study reported that a library of eight ASFV genes, including B646L (p72), CP204L (p30), CP530R (pp62), MGF 110-4 L, and 110–5, stocked with replication-deficient human adenovirus five progeny and a modified cowpox Ankara vaccine booster resulted in a percentage of pigs infected with the OUR T88/1 isolate that showed a slowing of clinical signs and reduced levels of viremia [168]. Follow-up studies of its immunization program showed it achieved 100% protection against fatal disease in pigs challenged with the OUR T88/1 strain [168]. In August 2023, the Guangzhou Institute of Biomedicine and Health (GIBH) of the Chinese Academy of Sciences (CAS) and the Guangzhou Laboratory, among others, constructed adenovirus-vectored African swine fever (ASF) vaccines carrying optimally designed combinations of multiple antigens, which have been shown to protect swine in the field [169]. A comparison of the advantages and disadvantages of various vaccines and immunization strategies is shown in Table 3.

Table 3.

Advantages and disadvantages of different vaccines for ASF and research progress.

Types of vaccines	Advantage	Disadvantage	Immunization strategy
Inactivated vaccine	Safe	antibody response production conditions necessitate strict	Mirasol Processing technique
Live attenuated vaccine	Targeted; Protective efficacy is significant; stimulation of cellular immunity	standards; The absence of passaged cell lines necessary for production; potential for homologous recombination and reversion of virulence	Utilizing porcine bone marrow and renal cell passage for attenuation; ASFV-G∆1177L;Lv17/WB?riel;NH/P68
Subunit vaccine	Safe, verifiable, can scalable production	need to enhance immunity and identify specific protective antigens	p54,p30,p72 and CD2v
DNA vaccine	Safe, verifiable, can scalable production	need to enhance immunity and identify specific protective antigens	p34/p30 funsion protein and CD8+
Viral vector vaccines	Safe, verifiable, can scalable production	need to enhance immunity and identify specific protective antigens	Our T88/1 strain; adenovirus vector-based vaccine for african swine fever

Progress in antiviral drug research

Nucleoside analogs block viral replication by inhibiting nucleic acid synthesis or enzymes involved in nucleoside metabolism. The first nucleoside analogue found to be active against ASFV was iododeoxyuridine, which at a concentration of 100 μg/mL, completely inhibited ASFV in Vero cells, resulting in a reduction of ASFV by about four logs [170,171]. The small-molecule nucleoside analog cidofovir (cHPMPC) inhibits the replication of different genotypes of ASFV in primary porcine alveolar macrophages [172]. cHPMPC effectively inhibited ASFV replication and viral gene expression when added before or early in the infection, thus surmising that the target of the drug may be the viral DNA polymerase or RNA polymerase [172]. Guo et al. found that Brincidofovir could inhibit ASFV replication in a dose-dependent manner through in vitro experiments. Brincidofovir could significantly reduce the viral titer of ASFV and inhibit the expression of viral structural proteins [173]. The results of the drug addition test showed that Brincidofovir acted on the post-entry phase of the virus. In vivo, experimental data showed that Brincidofovir could effectively inhibit the replication and horizontal transmission of ASFV in pigs [173]. ASFV-infected piglets were orally administered Brincidofovir, which resulted in a significant increase in survival and a significant decrease in hyperthermia, viremia, detoxification, and histopathological damage caused by the infection [173]. This study is the first to identify and validate the inhibitory activity of Brincidofovir against ASFV in vitro and in vivo, providing new ideas and strategies for developing drugs for the prevention and control of African swine fever.

Natural products are compounds, and their metabolites are found in various plants and animals. In recent years, they have been extensively studied for their antiviral properties. Lignans are widely found in onions, carrots, broccoli, and apples and have anticancer and antiviral biological activities [174]. Studies have shown that lignans can inhibit ASFV replication by modulating the NF-κB/STAT3/ATF6 signaling pathway [174]. Flavonoids or polyphenols are secondary metabolites present in plants such as vegetables, fruits, seeds, etc., and they have a wide range of biological activities, including anti-inflammatory, anticancer, and anti-infective. According to literature reports, several naturally occurring plant flavonoids, including goldfinch isoflavones, apigenin, and kaempferol, have been found to have inhibitory effects on ASFV. Goldfinch isoflavones inhibit ASFV type II topoisomerase, blocking viral DNA replication and protein synthesis [175]. Apigenin has a potent, dose-dependent inhibitory effect on ASFV replication in Vero cells, but it is insoluble in highly polar solvents and usually occurs as a derivative in plants [176]. Therefore, the anti-ASFV effect of derivatives of apigenin was investigated. It was found that coriander reduced the levels of early and late ASFV proteins, as well as the synthesis of viral DNA, and was also able to inhibit ASFV invasion and outgrowth, probably by hindering the transport of ASFV with the help of microtubules [175]. Berbamine hydrochloride, a bis benzylisoquinoline alkaloid isolated from the traditional Chinese herb Berberis vulgaris, effectively inhibited ASFV replication in cells in a dose-dependent manner [177]. In addition, many other natural products and their derivatives, some marine algal extracts, including resveratrol and lauryl gallate, have been reported to inhibit ASFV [178–181].

Inhibitors directly targeting viruses mainly exert antiviral effects by directly acting on viral proteins or genomes. Currently, the most researched inhibitors target the ASFV protein pS273R. Liu et al. obtained a group of small molecule compounds by molecular docking screening based on the structural information of pS273R. Among them, E-64 can effectively inhibit the enzyme activation center of the pS273R protease, prevent pS273R protease from cleavage of pp62, and promote the upregulation of immune-associated cytokines at the transcriptional level [182]. In addition, Liu et al. resolved the high-resolution structure of the African swine fever virus capsid protein p72 for the first time by using cryo-electron microscopy single-particle three-dimensional reconstruction, revealing the possible assembly mechanism of the African swine fever virus capsid, which laid a solid foundation for the development of the subunit vaccine of African swine fever virus [183]. Zhao et al. resolved the cryo-electron microscopy structure of P1192R in two different functional states, making progress in the structural resolution and catalytic mechanism of African swine fever viral topoisomerase, enhancing scientists’ understanding of the structure and function of viral topoisomerase and providing an essential basis for the research and development of anti-African swine fever drugs targeting viral topoisomerase [184].

Other measures

Prevention and control of swine fever in pig farms

The location of pig farms should be in strict accordance with the requirements, built in a high and flat dry place, far away from domestic drinking water sources, and residential areas more than 500 meters [185]. It should be downwind to ensure that the production process of the farms does not pollute the surrounding residents. Farming is prohibited in the prohibited area, and neighboring pig farms should be spaced far enough apart from each other to avoid mutual pollution [185]. Fences should be enclosed on pig farms to prevent outsiders from entering, and living and production areas should be divided. Disinfection should be noted when entering and leaving the farm.

Establishment of monitoring and reporting mechanism

Establish a sound surveillance system, including formulating a surveillance plan, establishing a surveillance network, and clarifying surveillance indicators and methods. Regularly monitor the health of pigs, check their body temperature, appetite, mental state, etc., and pay close attention to the spread and changes of the epidemic. For suspected cases or abnormalities found, they should be reported to the relevant departments promptly to ensure the timely and accurate transmission of epidemic information. At the same time, it is necessary to strengthen the investigation and monitoring of the means of transmission and the scope of influence of the epidemic so that changes and trends can be detected promptly. Training has been provided to staff involved in surveillance to improve their understanding of African swine fever and their surveillance skills, to ensure the accuracy and timeliness of surveillance, and to provide a reference for subsequent work on managing the epidemic [186].

Isolation and disinfection measures

Isolation is critical for preventing and controlling African swine fever [186]. The imported pigs should be isolated and fed in time. It is recommended to use the independent enclosure for isolation after the sterilization of empty pens, the isolation house should be in the downwind direction of other barns, and the nasal and anus swabs should be collected during the isolation period to detect African swine fever, to ensure that there are no abnormalities during the isolation period. The pigs can be mixed and reared after the period. We should observe the health status of pigs promptly and take measures such as isolation treatment or elimination treatment for pigs found in abnormal conditions. Pigs should be imported from a single source, and importing pigs (including breeding pigs) from different farms is not recommended [186].

In addition, it is necessary to do a good job of disinfection and disposal [187]. First, the entire area should be disinfected, and the frequency can generally be determined according to the environment and the incidence of the disease in the area. The office or living area’s roof, walls, and floor can be disinfected by spraying aldehyde or chlorine disinfectant. The floor of the field or compound can be disinfected by spraying aldehydes and alkaline disinfectant solution or lime slurry bleach. The basic steps in pen disinfection are cleaning, washing, disinfecting, and secondary disinfection. Removing items from the pens, removing debris, and cleaning and treating are essential. When cleaning, use a low-pressure water gun to soak the floor, rails, and other parts to an utterly wet condition, then use a foam gun to spray cleaners, then use a high-pressure water gun to rinse and use hot water to rinse if possible, from top to bottom, from inside to outside. After drying, aldehyde and peroxide disinfectants were used to spray disinfection for 1 h after the second disinfection. If the house is a closed environment, fumigation can also be used for disinfection [187,188].

The vehicle disinfection process consists of five steps: washing, inspection, disinfection, drying, and effectiveness evaluation. A vehicle washroom should be set up outside the pig farm. Vehicles should be cleaned and processed before disinfection, and when cleaning the cab, focus on the steering wheel, pedals, seats, doors, etc. When cleaning the body, tires, and compartments, concentrate on removing pig manure, urine, hair, and other contaminants. Disinfection in the cab can be taken to wipe the method of disinfection, the body to spray disinfectant way to disinfect, spray disinfectant should be static for about 20 min, and then rinse. Then, the disinfected vehicle is dried. Finally, swab samples can be taken from different parts of the car for rapid testing to assess the effect, and a negative test is considered qualified for disinfection [188,189].

Discussion

African swine fever (ASF) is one of the significant epidemics currently threatening the sustainable development of the global swine industry, posing a considerable threat and obstacle to pig production and socio-economic stability. The knowledge of ASFV is still extremely limited due to its large genome, complex structure, the unknown function of many genes, and the influence of various factors such as genotypic complexity, immune escape mechanism, immune protection mechanism, etc. Therefore, it is necessary to strengthen the research on the pathogenesis and epidemiology of ASFV to reveal the pattern of the genetic evolution of virulent strains, transmission characteristics, and pathogenicity. There have been many studies on vaccines against ASFV, but few in-depth studies on protective mechanisms have been reported. Zhu et al. analyzed some possible mechanisms of ASFV pathogenesis and immune escape through gene expression changes in ASFV-infected macrophages [68]. Sun et al. first developed an ASF cellular immune vaccine by panoramic scanning of T-cell epitopes of p72 proteins [190]. Therefore, deep sequencing and bioinformatics analysis studies based on transcriptome and proteome will help to resolve the information of the core genes and key antigens of ASFV immune escape, which is crucial for subsequent immune escape and vaccine development.

The review provides a more detailed and comprehensive overview of the latest research progress on the structure and function, biological properties, genome, transcriptome, proteome, transmission pathways, and preventive and control measures of ASFV in the hope that it will help to deeply understand the pathogenic mechanism of ASFV and molecular mechanism of immune escape, and provide theoretical support for the research and development of ASF vaccine and disease prevention and control.

Funding Statement

This work was supported by grants from the National Center of Technology Innovation for Pigs (NCTIP-XD/C03) project, which is undertaken by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization, Mei Li and Haixue Zheng; data curation, Mei Li and Haixue Zheng; writing original draft preparation, Mei Li; writing review and editing, Haixue Zheng; funding acquisition, Haixue Zheng. All authors have read and agreed to be published.

Data availability statement

Data sharing does not apply to this article, as no new data was created or analyzed.