Morphological and structural characteristics of ASFV

ASFV is a large, complex double-stranded DNA arbovirus and a member of the Asfarviridae family (Blome et al., Reference Blome, Franzke and Beer2020). ASFV has a complex multilayered structure with an overall icosahedral morphology (Fig. 1), and the average diameter of the particles is 260–300 nm. ASFV can be divided into five layers from the inside to the outside (Fig. 2). The innermost layer is the central genomic nucleolus, and the second layer is the thick protein core shell covering the nucleolus. The third layer is the inner lipid membrane. The fourth layer is an icosahedral protein capsid, and ASFV acquires its outermost envelope, the fifth layer, when budding through the host plasma membrane. The core-shell diameter is 180 nm, and it is covered by a 70 Å-thick lipid bilayer membrane (Wang et al., Reference Wang, Zhao, Wang, Zhang, Wang, Gao, Li, Wang, Bu, Rao and Wang2019). The maximum diameter of the main structural capsid of ASFV is 250 nm, and its structure has been solved at a resolution of 4.1 Å. The capsid structure consists of multiple proteins, including one major capsid protein (p72) and four minor proteins (M1249L, p17, p49, and H240R). These capsid proteins consist of 12 five-symmetrical and 20 three-symmetrical structures. Small capsid proteins (p17, p49, and M1249L) form a complex network beneath the outer shell. The capsid is fixed by adjacent proteins that assemble together to maintain its stability. Three 100 nm-long M1249L proteins run along each edge, connecting two adjacent five- and three-symmetrical regions and forming an extensive intermolecular network with other capsid proteins to enable the formation of the capsid skeleton (Alejo et al., Reference Alejo, Matamoros, Guerra and Andres2018; Liu et al., Reference Liu, Ma, Qian, Zhang, Tan, Lei and Xiang2019b).

Figure 1. Morphological structure of ASFV (Liu et al., Reference Liu, Ma, Qian, Zhang, Tan, Lei and Xiang2019b). (Reproduced directly from Liu et al., Reference Liu, Ma, Qian, Zhang, Tan, Lei and Xiang2019b Cell Host Microbe with permission from the Cell Press).

Figure 2. Schematic diagram of ASFV’s structure.

Clinical symptoms and pathological changes upon ASFV infection

According to the virulence of the virus, infection is divided into the following categories based on clinical symptoms: severe acute, acute, subacute, chronic, and atypical course (Avagyan et al., Reference Avagyan, Hakobyan, Baghdasaryan, Arzumanyan, Poghosyan, Bayramyan, Semerjyan, Sargsyan, Voskanyan, Vardanyan, Karalyan, Hakobyan, Abroyan, Avetisyan, Karalova, Semerjyan and Karalyan2024; Sanchez-Cordon et al., Reference Sanchez-Cordon, Floyd, Hicks, Crooke, McCleary, McCarthy, Strong, Dixon, Neimanis, Wikstrom-Lassa, Gavier-Widen and Nunez2021; Wang et al., Reference Wang, Zhang, Hou, Yang and Wen2021b). Severe acute infection is caused by a highly virulent strain, which leads to the sudden death of pigs without clinical symptoms, resulting in a 100% mortality rate. Within 3–4 days after infection, pigs with acute disease typically exhibit a high fever, haemorrhaging of the reticuloendothelial system, loss of appetite and lethargy, and dyskinesia. The lymph nodes are usually swollen, fragile, and bleeding in post-mortem examinations (Sanchez-Cordon et al., Reference Sanchez-Cordon, Floyd, Hicks, Crooke, McCleary, McCarthy, Strong, Dixon, Neimanis, Wikstrom-Lassa, Gavier-Widen and Nunez2021). Some pigs with acute disease exhibit lymph node congestion, digestive problems, including gastrointestinal tract haemorrhage and oedema; central nervous system oedema; and perivascular haemorrhage. Within 4–15 days after infection, the case fatality rate is nearly 100% (Sanchez-Cordon et al., Reference Sanchez-Cordon, Floyd, Hicks, Crooke, McCleary, McCarthy, Strong, Dixon, Neimanis, Wikstrom-Lassa, Gavier-Widen and Nunez2021; Wang et al., Reference Wang, Zhang, Hou, Yang and Wen2021b). Medium-virulence strain infection causes a subacute infection, characterized by mild symptoms, primarily manifesting as respiratory distress, joint pain, and swelling, with low mortality (30–70%). Abortion may result from ASFV infection in pregnant sows. Low-virulence isolates can cause chronic symptoms, such as faeces containing mucus, miscarriage, vomiting, diarrhoea, bleeding, breathing changes, and other symptoms, but the mortality rate is low (Sánchez-Cordón et al., Reference Sánchez-Cordón, Montoya, Reis and Dixon2018). The incubation period of ASFV generally ranges from 5 to 15 days, with some individual cases extending to 28 days. The atypical course has emerged in recent years, caused by lower virulent strains of ASFV, characterized by a significantly prolonged disease duration, sometimes reduced mortality, prolonged viremia, and no apparent clinical symptoms (Avagyan et al., Reference Avagyan, Hakobyan, Baghdasaryan, Arzumanyan, Poghosyan, Bayramyan, Semerjyan, Sargsyan, Voskanyan, Vardanyan, Karalyan, Hakobyan, Abroyan, Avetisyan, Karalova, Semerjyan and Karalyan2024; Sun et al. Reference Sun, Zhang, Wang, He, Zhang, Wang, Wang, Huang, Xi, Huangfu, Tsegay, Huo, Sun, Tian, Xia, Yu, Li, Liu, Guan, Zhao and Bu2021).

ASFV is highly stable in the environment; therefore, it can remain infectious for several days in faeces or urine under suitable conditions and can survive even longer in organic matter (Mazur-Panasiuk et al., Reference Mazur-Panasiuk, Zmudzki and Wozniakowski2019). In the secretions, excrement, blood, and tissues of infected pigs, its activity can be maintained for several weeks, and it can also hold its activity for a long time in smoked and air-dried foods, such as cured dry ham, in which it can survive for 150 days. ASFV is also highly resistant to acidic and alkaline conditions, and the virus can remain stable at pH values ranging from 3.9 to 11.5. The virus is resistant to low temperatures but is sensitive to high temperatures. It can survive for 30 min at 55 ℃, 10 min at 60 ℃, several weeks at 25–37 ℃, and more than 1 year at 4 ℃. Additionally, it can survive in frozen pork up to 2 years (Mazur-Panasiuk and Woźniakowski, Reference Mazur-Panasiuk and Woźniakowski2020). Numerous studies have demonstrated the efficacy of various lipid solvents, detergents, and disinfectants containing aldehyde-, phenol-, or iodide-based compounds in inactivating ASFV (Juszkiewicz et al., Reference Juszkiewicz, Walczak, Wozniakowski, Pejsak and Podgorska2025; Ni et al., Reference Ni, Chen, Yun, Xie, Ye, Hua, Zhu and Zhang2023). In particular, commercially available disinfectants such as compound peroxymonosulfate, sodium dichloroisocyanurate, and glutaraldehyde have been widely implemented in biosecurity protocols, especially in China, where ASF outbreaks have been most prevalent (Juszkiewicz et al., Reference Juszkiewicz, Walczak, Mazur-Panasiuk and Woźniakowski2020). These compounds have shown significant virucidal activity against ASFV in both laboratory and field conditions.

Epidemiological characteristics of ASFV

ASF was first observed in settlers’ pigs in Kenya, Africa, in 1909 and was first reported in 1921 as a distinct disease from classical swine fever (Montgomery, Reference Montgomery1921). Reports of ASF in South Africa and Angola (Steyn, 1932) followed. ASF broke out in the Lisbon area of Portugal In 1957 and again in 1960 (Wilkinson, Reference Wilkinson and Pensaert1989); then, it spread to the Iberian Peninsula and spread further in Europe as well as in the Caribbean and Brazil in South America (Costard et al., Reference Costard, Wieland, de Glanville, Jori, Rowlands, Vosloo, Roger, Pfeiffer and Dixon2009). During the same time, ASF was also found in Malawi and Mozambique (Abreu et al., Reference Abreu, de Valadão, Limpo Serra, Ornelas Mário and Sousa Montenegro1962; Matson, Reference Matson1960; Mendes, Reference Mendes1971). Researchers have shown that some intermediate animals, such as warthogs (Phacochoerus africanus) and Ornithodoros moubata complex ticks, were associated with virus ASFV infection and transmission at that time in sub-Saharan Africa (Montgomery, Reference Montgomery1921; Penrith et al., Reference Penrith, Vosloo, Jori and Bastos2013). ASFV strains are currently grouped into 24 genotypes based on the reference B646 L gene (p72), and all the gene variants are associated with the disease (Quembo et al., Reference Quembo, Jori, Vosloo and Heath2018). The 24 genotypes are clustered into three distinct evolutionary lineages, each confined to a broad geographical area. Lineage I comprised 14 genotypes (including I, XVII, II, XXIV, XXI, V, VI, XVIII, VII, XXII, IV, III, XX, and XIX) associated with viruses from West and southern Africa. In contrast, lineage II (including XIV, XVI, XV, XIII, XII, VIII, and XI) consisted of viruses from East Africa and lineage III (including X, IX, and XIII) consisted of viruses from the Great Lakes Region of East and Central Africa (Bastos et al., Reference Bastos, Penrith, Cruciere, Edrich, Hutchings, Roger, Couacy-Hymann and RT2003; Boshoff et al., Reference Boshoff, Bastos, Dube and Heath2014; Quembo et al., Reference Quembo, Jori, Vosloo and Heath2018). Most of these genotypes have been associated with ASF outbreaks in various parts of sub-Saharan Africa (Mulumba-Mfumu et al., Reference Mulumba-Mfumu, Saegerman, Dixon, Madimba, Kazadi, Mukalakata, Oura, Chenais, Masembe, Stahl, Thiry and Penrith2019). Genotype I has been shown to predominate in Central Africa (Kouakou et al., Reference Kouakou, Michaud, Biego, Gnabro, Kouakou, Mossoun, Awuni, Minoungou, Aplogan, Awoume, Albina, Lancelot and Couacy-Hymann2017) and West Africa (Hakizimana et al., Reference Hakizimana, Nyabongo, Ntirandekura, Yona, Ntakirutimana, Kamana, Nauwynck and Misinzo2020). Most Tanzanian ASF outbreaks have been linked to genotypes II, IX, X, XV, and XVI of the virus (Misinzo et al., Reference Misinzo, Magambo, Masambu, Yongolo, Van Doorsselaere and Nauwynck2011; Njau et al., Reference Njau, Machuka, Cleaveland, Shirima, Kusiluka, Okoth and Pelle2021), while in other areas in Mauritius, Madagascar, Tanzania, and Zimbabwe, the prevalent highly virulent strain of ASFV has been identified as genotype II (Wambura et al., Reference Wambura, Masambu and Msami2006). In addition, genotypes IX and X were identified in Uganda and Kenya (Atuhaire et al., Reference Atuhaire, Afayoa, Ochwo, Mwesigwa, Okuni, Olaho-Mukani and Ojok2013; Gallardo et al., Reference Gallardo, Okoth, Pelayo, Anchuelo, Martin, Simon, Llorente, Nieto, Soler, Martin, Arias and Bishop2011). From 1968 to 1995, ASFV of the p72 genotype I was present in European countries, including Malta, Sardinia, Italy, France, Belgium, and the Netherlands. The prevalent genotype in the Gulu district is genotype IX (Barongo et al., Reference Barongo, Stahl, Bett, Bishop, Fevre, Aliro, Okoth, Masembe, Knobel and Ssematimba2015). ASF was first reported in Cuba in North America in 1971, after which ASF epidemics caused by ASFV of genotype I also occurred in Brazil, the Dominican Republic, and other countries (Ankhanbaatar et al., Reference Ankhanbaatar, Auer, Ulziibat, Settypalli, Gombo-Ochir, Basan, Takemura, Tseren-Ochir, Ouled Ahmed, Meki, Datta, Soumare, Metlin, Cattoli and Lamien2023). By the mid-1990s, ASFV had been eradicated outside Africa, except for an isolated outbreak in Portugal in 1999 and an infection on the island of Sardinia (Italy) (p72 genotype I) (Boinas et al., Reference Boinas, Wilson, Hutchings, Martins and Dixon2011; Laddomada et al., Reference Laddomada, Rolesu, Loi, Cappai, Oggiano, Madrau, Sanna, Pilo, Bandino, Brundu, Cherchi, Masala, Marongiu, Bitti, Desini, Floris, Mundula, Carboni, Pittau, Feliziani, Sanchez-Vizcaino, Jurado, Guberti, Chessa, Muzzeddu, Sardo, Borrello, Mulas, Salis, Zinzula, Piredda, De Martini and Sgarangella2019; Pavone et al., Reference Pavone, Iscaro, Dettori and Feliziani2023). After a new introduction of the virus from Africa into Georgia in 2007, ASFV genotype II spread to the Caucasus and reached the Russian Federation (Beltrán-Alcrudo et al., Reference Beltrán-Alcrudo, Lubroth, Depner and De La Rocque2008). It then spread further from Russia to the European Union in 2014 (Dixon et al., Reference Dixon, Stahl, Jori, Vial and Pfeiffer2020). In August 2018, the virus reached China, the world’s largest pig-producing country, and is currently spreading in most of Southeast Asia and Oceania (Hakizimana et al., Reference Hakizimana, Nyabongo, Ntirandekura, Yona, Ntakirutimana, Kamana, Nauwynck and Misinzo2020; Mulumba-Mfumu et al., Reference Mulumba-Mfumu, Saegerman, Dixon, Madimba, Kazadi, Mukalakata, Oura, Chenais, Masembe, Stahl, Thiry and Penrith2019; Njau et al., Reference Njau, Machuka, Cleaveland, Shirima, Kusiluka, Okoth and Pelle2021; Quembo et al., Reference Quembo, Jori, Vosloo and Heath2018; Urbano and Ferreira, Reference Urbano and Ferreira2022). Since then, the epidemiological situation of ASF has continued to deteriorate. In July 2021, after nearly 40 years of absence, ASF was reintroduced in the Dominican Republic and later in Haiti. In January 2022, it reappeared on the Italian mainland (Urbano and Ferreira, Reference Urbano and Ferreira2022). Although ASFV genotype II is currently the dominant strain, genotype I has also been identified in China (Cao et al., Reference Cao, Lu, Wu and Zhu2022). Furthermore, some highly lethal genotype I and II recombinant ASFV strains have been detected in pigs (Zhao et al., Reference Zhao, Sun, Huang, Ding, Zhu, Zhang, Shen, Zhang, Zhang, Ren, Wang, Li, He and Bu2023). The new defined genotype XXIV was identified in soft ticks from Gorongosa National Park in Africa (Quembo et al., Reference Quembo, Jori, Vosloo and Heath2018). The World Animal Health Information System of the World Organization for Animal Health (WOAH) has released a map of the real-time global distribution of ASF (Fig. 3) (https://wahis.woah.org/#/event-management), from which it is evident that ASF is spreading rapidly in Africa, Asia, Europe, and other regions. Research on ASF prevention, control, and treatment is therefore urgent. In addition to wild pigs and soft ticks, the natural hosts of ASFV, the virus can also spread through direct or indirect contact with infected pigs and their products. The ASFV transmission cycle mainly includes the sylvatic cycle (wild boar–soft tick–wild boar cycle), wild boar–domestic pig cycle, and domestic pig–domestic pig cycle (Karger et al., Reference Karger, Pérez-Núñez, Urquiza, Hinojar, Alonso, Freitas, Revilla, Le Potier and Montoya2019). Given the above, vigilance and continuous surveillance will be crucial while the quest for an ASF vaccine continues.

Note: The number on the map represents the number of outbreaks of ASF that occurred in the area from 3 October 2018 to 29 June 2025.

Figure 3. Global distribution of ASFV as of 29 June 2025.

In the sylvatic cycle (wild boar–soft tick–wild boar), soft ticks play an important role in transmission between wild boars, and ASFV can be transmitted between different soft ticks. After a soft tick bites an infected wild boar, the virus can survive in the soft tick for a long time and then enter the next cycle when the tick bites another wild boar. The blood, excreta, and secretions of infected wild pigs contain high levels of the virus. Therefore, through direct or indirect contact with infected pigs or contaminated surfaces, feed, and water, ASFV spreads rapidly between wild pigs, leading to long-term stability of the sylvatic cycle (Mazur-Panasiuk et al., Reference Mazur-Panasiuk, Zmudzki and Wozniakowski2019). Wild boar–domestic pig cycle: ASFV spreads rapidly in wild boar herds; this virus can cross borders through the wildlife corridor and become a source of infection in domestic pigs. In regions with high wild boar densities, direct or indirect contact with domestic pigs, particularly in low-biosecurity settings where pigs are allowed to forage outdoors, facilitates the transmission of viral pathogens to domestic populations. ASFV spreads among domestic pigs primarily through direct contact with infected individuals, with transmission occurring via the oronasal route or skin abrasions upon exposure to blood, excreta, or secretions. Notably, pigs recovering from infection with moderately or low-virulent strains may become chronic carriers, perpetuating viral circulation within herds. Human-mediated transmission further exacerbates spread through fomites, including contaminated clothing, footwear, vehicles, equipment, and slaughter tools. Viral dissemination via contaminated pork products, swill, feed, faeces, and bedding can result in both localized and long-distance transmission, including cross-border spread (Golnar et al., Reference Golnar, Martin, Wormington, Kading, Teel, Hamer and Hamer2019; Niederwerder et al., Reference Niederwerder, Stoian, Rowland, Dritz, Petrovan, Constance, Gebhardt, Olcha, Jones, Woodworth, Fang, Liang and Hefley2019).

Nucleocapsid protein

There are 110 highly conserved ORFs in the ASFV genome, two of which encode the ASFV polyprotein precursors pp220 and pp62 (or pp60). The mature particles form after these two precursor proteins are hydrolysed and processed to assemble the core-shell of the virus (Liu et al., Reference Liu, Ma, Qian, Zhang, Tan, Lei and Xiang2019b).

The pp220 protein encoded by the CP2475 L gene has a molecular weight of 281.5 kDa and is participates in late-stage viral infection. As a protein scaffold, polyprotein pp220 can bind the nucleocapsid to the inner membrane of the virus and promote the assembly of the empty nucleocapsid. After polyprotein pp220 is hydrolysed and processed by the virus-encoded SUMO-like protease S273 R, it is converted into the mature virus particle proteins p150, p37, p34, p14, and the recently identified p5 (Andres et al., Reference Andres, Charro, Matamoros, Dillard and Abrescia2020). The relative molecular weight of the polyprotein pp62 encoded by the CP530R gene is 60.5 kDa, and this protein plays an important role in the replication, assembly, and maturation of ASFV. Upon hydrolysis by the S273R protease, pp62 is converted into the mature virus particle proteins p35 and p15 (Fu et al., Reference Fu, Zhao, Zhang, Zhang, Li, Zhang, Wang, Sun, Jiao, Chen, Guo and Rao2020), and the recently identified p8 (Alejo et al., Reference Alejo, Matamoros, Guerra and Andres2018). The mature virus particle proteins p150, p37, p14, p34, and p35 (Li et al., Reference Li, Fu, Zhang, Zhao, Li, Geng, Sun, Wang, Chen, Jiao, Cao, Guo and Rao2020) and p15 account for approximately 30% of the total viral protein and constitute the main components of the virus nucleocapsid. The p37 transport protein plays an important role in the ASFV replication cycle. The p34 protein has a protective effect on trypsin. The p150 protein is a component of the membrane. p5 and p8 are structural components of the ASFV virus particles, and with other polymer proteins, they are packed into the core of a virus particle (Alejo et al., Reference Alejo, Matamoros, Guerra and Andres2018).

Capsid proteins

The p72 protein is the main structural protein of the capsid, with a relative molecular mass of 73.2 kDa, and is the key antigen protein encoded by the B646L (VP72) gene (Liu et al., Reference Liu, Ma, Qian, Zhang, Tan, Lei and Xiang2019a). Newly synthesized p72 is evenly distributed among the soluble cytoplasm, the membrane, and the endoplasmic reticulum, where it accumulates to form a large capsid or matrix precursor. p72 has a highly conserved sequence with good antigenicity and immunogenicity; however, the anti-p72 antibody does not play a decisive role in antibody-mediated immune protection (Gallagher and Harris, Reference Gallagher and Harris2020).

The p49 protein is encoded by the B438L gene and is an essential structural protein for the formation of infectious virus particles (Dixon et al., Reference Dixon, Chapman, Netherton and Upton2013). The gene ORF consists of 1317 nucleotides, encodes 438 amino acids, and is expressed in viral factories. Upon p49 binding to the membrane, other proteins are recruited to the inner viral membrane to initiate assembly into a complete capsid. In the absence of the p49 protein, the virus particle has an abnormal tubular structure instead of a complete icosahedral symmetric structure.

The p14.5 protein, encoded by the E120R gene, and also known as the pE120R protein, has a molecular weight of 13.6 kDa. As a DNA-binding protein, p14.5 plays an important role in the formation of the core and capsid of the virus. It participates in the intracellular transport of virus particles. It is essential for the transfer of virus particles from the viral factories, where virus particles assemble in discrete cytoplasmic areas near the nucleus, to the plasma membrane of the cells (Andres et al., Reference Andres, Alejo, Salas and Salas2002; Jia et al., Reference Jia, Ou, Pejsak, Zhang and Zhang2017). pE120R is reportedly involved in regulating ASFV immune evasion, which inhibits the cGAS-STING-mediated immune response (He et al., Reference He, Yuan, Ma, Zhao, Yang, Zhang, Han, Wan and Zhang2022).

Inner envelope proteins

p54, encoded by the E183L gene, is also a critical ASFV antigenic structural protein with a relative molecular mass of 25 kDa. The protein is located in the outer lipid membrane of the virus particle. This protein can recruit the envelope precursor to the assembly site and plays a crucial role in virus adsorption and invasion, as well as in the inoculation of attenuated strains into pigs to induce specific antibodies (Rodríguez et al., Reference Rodríguez, García-Escudero, Salas and Andrés2004). Studies have shown that the p54 protein can stimulate the body to produce antibodies against this protein (Neilan et al., Reference Neilan, Zsak, Lu, Burrage, Kutish and Rock2004). In the early stage of the virus infection cycle, p54 protein inhibitors can suppress the activity of proteins related to virus attachment. Transient p54 expression in Vero cells demonstrated that this protein can activate caspase-3 and caspase-9, mediating cell apoptosis (Wang et al., Reference Wang, Zhang, Hou, Yang and Wen2021a).

p30 is encoded by the CP204 L gene and has a molecular mass of 30 kDa. The expression of the p30 protein indicates that the ASFV virus has entered the cell and is in the early stages of infection. The yeast two-hybrid system was used to screen for cellular proteins in the porcine macrophage cDNA library that may interact with the p30 protein, and heterogeneous ribonucleoprotein K (hnRNP-K) was identified as the most likely cellular ligand for the p30 protein (Hernaez et al., Reference Hernaez, Escribano and Alonso2008). It is now generally accepted that the p30 protein is one of the primary antigenic structural proteins in ASFV, and p30 has been identified as a subunit vaccine candidate (Petrovan et al., Reference Petrovan, Yuan, Li, Shang, Murgia, Misra, Rowland and Fang2019).

The pE248R protein, encoded by the E248R gene, is a crucial internal envelope protein involved in forming disulfide bonds (Alejo et al., Reference Alejo, Matamoros, Guerra and Andres2018). Studies have shown that the adsorption and internalization of the virus are not affected by the lack of the pE248R protein. Deletion of the pE248R protein inhibits the replication of virus particles in host cells, as well as the early and late expression of viral genes.

p17 is encoded by the ORF in the D117L gene, has a molecular weight of 13.1 kDa, and is a transmembrane protein (Xia et al., Reference Xia, Wang, Liu, Shao, Ao, Xu, Jiang, Luo, Zhang, Chen, Meurens, Zheng and Zhu2020). This protein facilitates the further assembly of the viral membrane precursor into an icosahedral intermediate, thereby increasing the viability of the virus. Blocking p17 expression inhibits the hydrolysis of the pp220 and pp62 proteins.

p12 is a membrane protein expressed in the late stage of ASFV infection, is encoded by an ORF in the O61 R gene, is 6.7 kDa in length, and is located in the inner envelope of the virus (Salas and Andrés, Reference Salas and Andrés2013). p12 plays an important role in ASFV attachment to susceptible cells. In addition to the abovementioned main proteins, p22, encoded by KP177L, and the pE199L, pH108R, and pH108R proteins play known roles, among which pE248R and pE199L are related to virus invasion.

Outer envelope protein

CD2v is the only characterized protein in the outer envelope and is encoded by an ORF in the EP402 R gene. It is also known as pEP402R and is similar to the T lymphocyte surface adhesion receptor CD2, with a relative molecular weight of 105 kDa. CD2v is a glycoprotein composed of a signal peptide, a transmembrane region, and two immunoglobulin-like domains, and it is located near the viral factories. Due to the presence of ligands on the surface of pig red blood cells, ASFV is easily internalized by red blood cells. Studies have shown that CD2v-mediated red blood cell adsorption facilitates the spread of the virus in the host (Burmakina et al., Reference Burmakina, Malogolovkin, Tulman, Xu, Delhon, Kolbasov and Rock2019). Proline repeats in the cytoplasmic region of CD2v can interact with the actin adaptor protein SH3P7, participating in vesicle transport and signal transduction (Rodríguez et al., Reference Rodríguez, Yáñez, Almazán, Viñuela and Rodriguez1993). The differences in the repeated sequences of different strains can be used to analyse the serotype of a strain. The CD2v protein exhibits good immunogenicity and is a choice for the development of cross-protective vaccines. Additionally, the CD2v protein can impair lymphocyte function and is involved in cell adhesion, enhancing virulence and immune regulation. CD2v also plays essential roles in ASFV tissue tropism, immune evasion, and the promotion of virus replication in the host (Malogolovkin et al., Reference Malogolovkin, Burmakina, Titov, Sereda, Gogin, Baryshnikova and Kolbasov2015).

Other proteins

The p10 protein is encoded by the K78R gene and has a relative molecular mass of 8.4 kDa. This protein participates in the adsorption of ASFV and can bind single-stranded or double-stranded DNA. Amino acids 71–73 of p10 play an important role in the introduction of virus particles into nuclei. The pA104 R protein may participate in the transcription, DNA replication, and genome packaging of ASFV (Alejo et al., Reference Alejo, Matamoros, Guerra and Andres2018).

The replication mechanism of ASFV

ASFV enters host cells through clathrin-mediated and dynein-dependent endocytosis, as well as macropinocytosis, including actin-dependent endocytosis and endocytosis involving microtubule activity (Andrés, Reference Andrés2017; Hernaez et al., Reference Hernaez, Guerra, Salas and Andres2016). ASFV can enter cells through a process known as classic phagocytosis, also referred to as receptor-mediated endocytosis. However, the cell receptors and viral ligands involved are currently unknown (Galindo et al., Reference Galindo, Cuesta-Geijo, Hlavova, Muñoz-Moreno, Barrado-Gil, Dominguez and Alonso2015).

The endocytosed virions are partially disrupted in late endosomes, where they lose their outer membrane and outer capsid. The capsid decomposes in the acidic endosomal lumen (Matamoros et al., Reference Matamoros, Alejo, Rodríguez, Hernáez, Guerra, Fraile-Ramos and Andrés2020). After capsid degradation and host membrane fusion mediated by the pE248R protein, the ASFV core is released into the cytoplasm (Hernaez et al., Reference Hernaez, Guerra, Salas and Andres2016). The core is transported through microtubules to the viral factories, where virus-encoded RNA polymerase and transcription factors replicate the virus. Approximately, 20% of the genes in ASFV are involved in transcription and mRNA modification. The newly generated virus particles are released from infected cells through a process known as budding. ASFV disrupts the structure of the subnuclear domain and chromatin, inducing changes in nuclear structure, facilitating the inhibitory nuclear environment, and enabling the virus to replicate efficiently in host cells (Sánchez et al., Reference Sánchez, Quintas, Nogal, Castelló and Revilla2013; Simões et al., Reference Simões, Freitas, Leitão, Martins and Ferreira2019); however, the full role of the nucleus in this process is still unclear. Therefore, further research is needed.

The mechanism of the host immune response to ASFV

Early experimental studies have demonstrated that immune serum and monoclonal antibodies from convalescent pigs can neutralize many ASFV isolates and that these antibodies can prevent the virus from attaching to cells in the early stage (Dixon et al., Reference Dixon, Islam, Nash and Reis2019). Recent experiments have demonstrated that antibodies specific to ASFV can provide immune protection, but cannot offer cross-protection (O’Donnell et al., Reference O’Donnell, Holinka, Krug, Gladue, Carlson, Sanford, Alfano, Kramer, Lu, Arzt, Reese, Carrillo, Risatti and Borca2015). Antibody neutralization is a sensitive and easily reversible process, and ASFV aggregates easily and cannot be recognized by specific antibodies. Therefore, antibody-mediated protection cannot completely neutralize the virus. Viruses that are not neutralized by antibodies can cause persistent infection in pigs (Neilan et al., Reference Neilan, Zsak, Lu, Burrage, Kutish and Rock2004). Antibodies may neutralize the virus in two ways: in one process, the virus binds to Vero cells or porcine alveolar macrophages, and through saturation binding, the antibody blocks the virus from attaching to the cell (Sánchez et al., Reference Sánchez, Pérez-Núñez and Revilla2017); in the other process, the ASFV neutralization-mediated mechanism is activated to inhibit virus internalization by Vero cells or porcine alveolar macrophages in the presence of immune serum. In summary, ASFV-specific antibodies partially inhibit the virus’s attachment but cannot provide complete protection (Escribano et al., Reference Escribano, Galindo and Alonso2013; Sunwoo et al., Reference Sunwoo, Pérez-Núñez, Morozov, Sánchez, Gaudreault, Trujillo, Mur, Nogal, Madden, Urbaniak, Kim, Ma, Revilla and Richt2019).

An increasing number of experiments have demonstrated that the cellular immune response mediated by cytotoxic T lymphocytes (CTLs) plays a crucial role in the process of ASFV infection (Argilaguet et al., Reference Argilaguet, Perez-Martin, Nofrarias, Gallardo, Accensi, Lacasta, Mora, Ballester, Galindo-Cardiel, Lopez-Soria, Escribano, Reche and Rodriguez2012; Bosch-Camós et al., Reference Bosch-Camós, López, Collado, Navas, Blanco-Fuertes, Pina-Pedrero, Accensi, Salas, Mundt, Nikolin and Rodríguez2021). The classical method of antigen presentation by major histocompatibility complex (MHC) class I molecules is related to the activation of cellular immunity (Rock and Shen, Reference Rock and Shen2005). The protein produced by ASFV replication and translation in the cell is degraded by the host cell’s proteasome to produce a series of short peptides. Some of these short peptides are transported to the endoplasmic reticulum by transport associated with antigen processing (TAP). The heavy chain and light chain β2 m microglobulin (β2 m) of class I molecules assemble to form a heterodimer, and the protein is then processed and modified by the Golgi apparatus to achieve correct spatial folding. Finally, the protein is transported to the cell membrane surface, where it binds to and activates the CD8⁺ T lymphocyte receptor (TCR), thereby inducing cellular immune function that can kill and eliminate target cells infected by ASFV (Gao et al., Reference Gao, He, Quan, Jiang, Lin, Chen and Qu2017).

Experiments have shown that the targets of this virus-specific CTL protein are usually limited to MHC class I antigens (Netherton et al., Reference Netherton, Goatley, Reis, Portugal, Nash, Morgan, Gault, Nieto, Norlin, Gallardo, Ho, Sánchez-Cordón, Taylor and Dixon2019). Blocking target cell antigens with monoclonal antibodies against MHC class I antigens (monoclonal antibodies, MAbs) significantly decreases CTL activity. The absence of CD8⁺ T lymphocytes results in a lack of specific cellular immune protection (Zhu et al., Reference Zhu, Ramanathan, Bishop, O’Donnell, Gladue and Borca2019). ASFV epitopes (p72, p32, p54, and CD2v) fused with ubiquitin serve as immune antigens; in the absence of specific antibodies, partial protection against lethal attacks is provided (Oura et al., Reference Oura, Denyer, Takamatsu and Parkhouse2005). The CD2v/p32/p54 fusion antigen, expressed by the baculovirus BacMam, also provides partial protection (Argilaguet et al., Reference Argilaguet, Pérez-Martín, López, Goethe, Escribano, Giesow, Keil and Rodríguez2013). An ASFV DNA expression library lacking CD2v, p32, and p54 has been used to develop vaccines that can partially protect animals from virulent strain attacks (Lacasta et al., Reference Lacasta, Ballester, Monteagudo, Rodriguez, Salas, Accensi, Pina-Pedrero, Bensaid, Argilaguet, Lopez-Soria, Hutet, Le Potier and Rodriguez2014). Pigs inoculated with attenuated ASFV strains are protected from the same or closely related virulent strains with a degree of cross-protection. The BA71ACD2 strain, with artificially deleted CD2v, can induce immune cross-protection against both homologous and heterologous viral strains, and can produce specific CD8⁺ T lymphocyte immunity (Burmakina et al., Reference Burmakina, Malogolovkin, Tulman, Xu, Delhon, Kolbasov and Rock2019).

Cytokines play a crucial role in activating cellular immunity, thereby influencing the treatment of ASFV infection (Salguero et al., Reference Salguero, Sánchez-Cordón, Núñez, Fernández de Marco and Gómez-Villamandos2005). For example, TNF-α is a pleiotropic cytokine that can inhibit viral infections, regulate the function of immune cells, and the production of molecules that mediate inflammatory responses (Barrado-Gil et al., Reference Barrado-Gil, Del Puerto, Galindo, Cuesta-Geijo, García-Dorival, de Motes and Alonso2021). The main source of TNF-α is macrophages, and TNF-α transcription is regulated by several transcription factors, such as NF-κB, NFAT, and c-Jun. Overexpression of these transcription coactivators can restore the activity of the TNF-α promoter. In addition to the effects of cytokines, different vectors expressing the same ASFV-specific antigen (e.g., poxvirus, adenovirus, pseudorabies virus) can also induce various degrees of immune response, and mixed immunizing cocktails can induce strong immune protection (Lokhandwala et al., Reference Lokhandwala, Waghela, Bray, Sangewar, Charendoff, Martin, Hassan, Koynarski, Gabbert, Burrage, Brake, Neilan and Mwangi2017; Maeda et al., Reference Maeda, West, Hayasaka, Ennis and Terajima2005). Therefore, the CTL-mediated cellular immune response plays an important protective role against ASFV infection. Future research on cellular immunity will also be beneficial for the production of effective subunit vaccines, as it eliminates the effect of ASFV infection on host cells and provides long-term protection. Therefore, CTLs play a crucial role in eliminating ASFV infection.

Repair mechanism of ASFV

When the virus invades, the host cell response causes DNA damage, leading to potentially lethal gene mutations in the viral genes or inhibition of the activity of viral DNA and RNA polymerases. ASFV utilizes the encoded DNA base excision repair (EBR) pathway enzyme to repair mutations, ensuring that the sequence is correctly copied (Redrejo-Rodríguez and Salas, Reference Redrejo-Rodríguez and Salas2014). The enzymes in the EBR pathway include DNA polymerase X repair, AP endonuclease, and DNA ligase. Additionally, the nucleotide-metabolizing enzyme and thymidine kinase encoded by ASFV are important for the effective replication of ASFV in macrophages. It is speculated that their actions increase the size of the dNTP library needed for viral genome replication (Redrejo-Rodríguez et al., Reference Redrejo-Rodríguez, Rodríguez, Suárez, Salas and Salas2013).

ASFV inhibits the type I interferon response

A series of proteins encoded by ASFV can inhibit the expression of type I interferons, cytokines, chemokines, adhesion molecules, interferon-stimulated genes (ISGs), and other immunoregulatory genes (Reis et al., Reference Reis, Abrams, Goatley, Netherton, Chapman, Sanchez-Cordon and Dixon2016). MGF360-15 R (A276R) inhibits IRF3 expression through a mechanism independent of the transcription factors IRF7 and NF-κB. The IRF3 receptor is part of a necessary pathway for activating the type I interferon response; therefore, the pathways activated by TLR3 and cytoplasmic sensing ultimately inhibit the induction of type I IFN. MGF505-7 R (A528R) inhibits the induction of type I IFN by inhibiting the transcription factors IRF3 and NF-κB, as well as inhibiting the type I and type II IFN signalling pathways (O’Donnell et al., Reference O’Donnell, Holinka, Sanford, Krug, Carlson, Pacheco, Reese, Risatti, Gladue and Borca2016). The ASFV pI329 L protein is similar to the cellular TLR3, a highly glycosylated protein expressed in the cell membrane. It can inhibit the TLR3-mediated induction of IFN-β and activation of NF-κB and IRF3. The pI329 L protein targets TRIF, and overexpression of TRIF can reverse the inhibitory effect of NF-κB and IRF3 activation (Zhu et al., Reference Zhu, Ramanathan, Bishop, O’Donnell, Gladue and Borca2019).

ASFV inhibits cell apoptosis

During viral entry and replication, ASFV inhibits the apoptosis and necrosis of the host cell through encoded proteins, ensuring that the virus has enough time for replication (Dixon et al., Reference Dixon, Sánchez-Cordón, Galindo and Alonso2017). The first early antiapoptotic protein encoded by the A179L gene is a homolog of Bcl-2 (Galindo et al., Reference Galindo, Hernaez, Díaz-Gil, Escribano and Alonso2008). The proapoptotic proteins in the BH3-only family include Bim, Bid, Puma, Noxa, Bmf, Bik, Bad, and Hrk. Bax and Bak are activated downstream of the BH3-only protein and play important regulatory roles in cell apoptosis. The A179L-encoded protein acts on upstream proapoptotic BH3 domain proteins (Bid, Bim, and Puma) and downstream Bak and Bax proteins to inhibit host cell apoptosis. Additionally, the A179L protein inhibits autophagy mediated by the BH3 domain of the autophagy regulator Beclin-only (Banjara et al., Reference Banjara, Caria, Dixon, Hinds and Kvansakul2017). The second late apoptosis inhibitor protein, A224L, is encoded by the A224L gene and is similar to the IAP gene-encoded apoptosis-like protein (IAP). A224L contains a single BIR motif at the NH2 end, and a 4-cysteine-type zinc finger domain is formed in the C-terminal region (Banjara et al., Reference Banjara, Shimmon, Dixon, Netherton, Hinds and Kvansakul2019). The expression of the A224L protein inhibited the apoptotic pathway involving caspase 3 and apoptosis induced by stimuli such as TNF-α signalling. The expression of the A224L protein can activate the NF-κB signalling pathway, enabling escape from host immunity and activating the transcription of many antiapoptotic genes, including IAP and Bcl-2 family members, to inhibit cell apoptosis (Portugal et al., Reference Portugal, Leitão and Martins2009).

Cell infection leads to a cellular stress response. As activated protein kinases phosphorylate translation initiation factors, eIF2-α reduces overall protein synthesis and targets CHOP, thereby activating the transcription of proapoptotic proteins. The ASFV DP71L protein interacts with PP1 and eIF-2α, thereby inducing the dephosphorylation of eIF-2α and inhibiting the activation of the proapoptotic transcription factor CHOP, which in turn inhibits the apoptosis induced by this pathway. pE153R contains a C-type lectin domain, and p53 inhibits cell apoptosis by activating the transcription of many apoptosis inhibitors (Hurtado et al., Reference Hurtado, Bustos, Granja, de León, Sabina, López-Viñas, Gómez-Puertas, Revilla and Carrascosa2011). Apoptosis can be observed in the later stages of infection because it may promote the spread of the virus through the apoptotic body and its subsequent absorption by monocytes/macrophages, thus preventing the inflammatory response caused by cell death through necrosis. Infected cells may secrete or present the cell surface factor TNF-α, which can induce the apoptosis of uninfected lymphocytes through the bystander effect. The large-scale destruction of lymphocytes inhibits the immune response mechanism.

The mechanism by which ASFV infection inhibits inflammation

ASFV has an elegant mechanism for evading the host immune defence system. ASFV can inhibit the secretion of IFN-α and TNF-α from macrophages, as well as the transcription of inflammatory cytokines, thereby reducing the level of mRNA transcription and translation (Golding et al., Reference Golding, Goatley, Goodbourn, Dixon, Taylor and Netherton2016). The pA238L protein encoded by the A238L gene is similar to the I-kB inhibitor of the NF-κB transcription factor and can inhibit NF-κB activation (Salguero et al., Reference Salguero, Gil, Revilla, Gallardo, Arias and Martins2008). In addition to activating the type I interferon promoter, the NF-κB transcription factor also transcriptionally activates many inflammatory responses. In resting cells, after calcineurin dephosphorylation, the NFAT factor is transferred to the nucleus to activate NFAT-dependent gene transcription. Meanwhile, pA238L binds to the calcium/calmodulin-regulated phosphatase calmodulin (CaN), inhibiting the pathway activated by the phosphatase and thus ultimately inhibiting the inflammatory response (Correia et al., Reference Correia, Ventura and Parkhouse2013).

Inactivated vaccines

Inactivated vaccines are a class of conventional vaccines that use physical or chemical methods to inactivate pathogenic microorganisms, causing them to lose their pathogenicity while retaining their antigenicity, which activates the body’s immune system upon immunization. Inactivated vaccines have the advantages of simple preparation and low cost. Research on inactivated ASF vaccines began in the 1960s; however, extensive studies have demonstrated that these vaccines primarily elicit humoral immunity, failing to induce robust cellular immune responses mediated by cytotoxic T cells. Consequently, they are unable to provide adequate protection against ASF (Cadenas-Fernandez et al., Reference Cadenas-Fernandez, Sanchez-Vizcaino, van den Born, Kosowska, van Kilsdonk, Fernandez-Pacheco, Gallardo, Arias and Barasona2021; Zhang et al., Reference Zhang, Zhao, Zhang, Qin, Shan and Cai2023). Although some adjuvants (e.g., Polygen, Emulsigen-D) or immune enhancers may enhance cellular immunity and increase vaccine efficacy in inactivated vaccines of other pathogens (Dar et al., Reference Dar, Kalaivanan, Sied, Mamo, Kishore, Suryanarayana and Kondabattula2013; Kaur et al., Reference Kaur, Saxena, Rai and Bhatnagar2010; Milian-Suazo et al., Reference Milian-Suazo, Gutierrez-Pabello, Bojorquez-Narvaez, Anaya-Escalera, Canto-Alarcon, Gonzalez-Enriquez and Campos-Guillen2011), inactivated ASF vaccines, when formulated with these adjuvants, continue to be insufficient to confer sterilizing immunity or durable immunoprotection (Blome et al., Reference Blome, Gabriel and Beer2014; Xie et al., Reference Xie, Liu, Di, Liu, Gong, Chen, Li, Yu, Lv, Zhong, Song, Liao, Song, Wang and Chen2022).

Attenuated vaccines

LAVs can be viruses attenuated through cell passage, naturally occurring ASFV strains with reduced virulence, or strains that are attenuated using gene-editing techniques to delete virulence factors (Krug et al., Reference Krug, Holinka, O’Donnell, Reese, Sanford, Fernandez-Sainz, Gladue, Arzt, Rodriguez, Risatti and Borca2015; Monteagudo et al., Reference Monteagudo, Lacasta, Lopez, Bosch, Collado, Pina-Pedrero, Correa-Fiz, Accensi, Navas, Vidal, Bustos, Rodriguez, Gallei, Nikolin, Salas and Rodriguez2017; Mulumba-Mfumu et al., Reference Mulumba-Mfumu, Goatley, Saegerman, Takamatsu and Dixon2016; Urbano and Ferreira, Reference Urbano and Ferreira2022).

Research on attenuated ASFV vaccines obtained by cell passage was first reported in 1957 (Detray, Reference Detray1957). However, previous research has shown that attenuated ASFV vaccines generated by cell passage can only provide homologous protection in animal challenge experiments (Manso-Ribeiro et al., Reference Manso-Ribeiro, Lopez-Frazao and Sobral1963). Spain and Portugal have isolated attenuated ASFV strains as vaccines; these strains provide homologous protection and partial heterologous protection, but vaccinated pigs exhibit various degrees of side-effects from the vaccine: a debilitating chronic disease showing viraemia and fever in a late phase of infection in many vaccinated pigs was caused because of these vaccines were insufficiently tested for their safety; as a result, the use of these vaccines was stopped (Gavier-Widen et al., Reference Gavier-Widen, Stahl and Dixon2020; Leitao et al., Reference Leitao, Cartaxeiro, Coelho, Cruz, Parkhouse, Portugal, Vigario and Martins2001). In 2015, an attenuated variant of the ASFV strain Stavropol 01/08 (Stavropol) isolated from the Russian Federation was obtained after 24 continuous passages in homologous pig kidney cells (A4C2/9k) and 20 passages in heterologous African green monkey CV-1 cells. The virus lost pathogenicity in pigs but did not protect them from death after challenge with a virulent virus, indicating that the attenuated Stavropol 01/08 could not provide enough protection for animals (Balysheva et al., Reference Balysheva, Galnbek and Balyshev2015). In the same year, Krug et al. (Krug et al., Reference Krug, Holinka, O’Donnell, Reese, Sanford, Fernandez-Sainz, Gladue, Arzt, Rodriguez, Risatti and Borca2015) reported the complete attenuation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) by 110 successive passages in Vero cells. However, immunization of swine with the fully attenuated virus did not confer protection against challenge with the virulent parental strain ASFV-G. Further detection revealed that amino acid substitutions and frameshift mutations occurred in the genome of the attenuated ASFV-G strain. According to Krug’s research, developing effective attenuated ASFV vaccines by cell passage is very difficult. Contemporaneously, Lacasta et al. (Reference Lacasta, Monteagudo, Jimenez-Marin, Accensi, Ballester, Argilaguet, Galindo-Cardiel, Segales, Salas, Dominguez, Moreno, Garrido and Rodriguez2015) reported that the cell culture-adapted strain E75CV1, obtained by adapting E75 to grow in the CV1 cell line, can potentially protect against homologous E75 virus challenge but was shown to have poor cross-protection against BA71; this finding indicated that E75CV1 provided partial heterologous protection against BA71. Recently, a novel naturally attenuated ASFV strain, ASFV-989, isolated from a blood sample of pigs inoculated with the thermo-attenuated ASFV Georgia 2007/1 strain, was reported by Bourry’s group to provide full clinical protection against challenge with Georgia 2007/1 through oronasal or intramuscular immunization (Bourry et al., Reference Bourry, Hutet, Le Dimna, Lucas, Blanchard, Chastagner, Paboeuf and Le Potier2022). ASFV-989 is a promising vaccine because it has not been genetically manipulated, which facilitates its use through open distribution in the wild, although a natural deletion of 7458 nucleotides located in the 5′ end encoding region of MGF 505/360, compared to that in the Georgia population (2007/1), was identified.

Another approach to developing attenuated vaccines is the screening of naturally attenuated, non-haem-adsorbing (non-HAD) strains and/or strains with reduced virulence that occur naturally during ASF epidemics (Urbano and Ferreira, Reference Urbano and Ferreira2022). Leitao et al. (Reference Leitao, Cartaxeiro, Coelho, Cruz, Parkhouse, Portugal, Vigario and Martins2001) reported a case in which a non-HAD, nonfatal genotype I ASFV strain, NH/P68, was isolated from a chronically infected pig and used to infect oronasally or intramuscularly to evaluate its effectiveness. The vaccinated pigs exhibited good cellular and humoral immunity and were protected against highly virulent ASFV/L60 challenge. Sanchez-Cordon et al. (Reference Sanchez-Cordon, Chapman, Jabbar, Reis, Goatley, Netherton, Taylor, Montoya and Dixon2017) also reported the naturally attenuated ASFV genotype I isolate OURT88/3, showing that intranasal immunization with low or moderate doses (10³ and 10⁴ TCID₅₀) of OURT88/3 provided complete protection (100%) against challenge with the virulent genotype I OURT88/1 isolate. Recently, Barasona et al. (Reference Barasona, Gallardo, Cadenas-Fernandez, Jurado, Rivera, Rodriguez-Bertos, Arias and Sanchez-Vizcaino2019) developed a non-HAD, attenuated ASF virus of genotype II isolated in Latvia in 2017 (Lv17/WB/Rie1), which conferred 92% protection against challenge with a virulent ASF virus isolate (Arm07). Gallardo et al. (Reference Gallardo, Soler, Rodze, Nieto, Cano-Gomez, Fernandez-Pinero and Arias2019) also confirmed that pigs intramuscularly infected with Lv/17/WB/Rie1 ASFV developed nonspecific clinical signs and, in some cases, remained asymptomatic, exhibiting intermittent and weak viremia, along with a high antibody response. Furthermore, 2 months following primary infection with Lv17/WB/Rie1, the two pigs were fully resistant to challenge with the virulent genotype II HAD Latvian ASFV. Although naturally attenuated strains have the potential to be developed as LAVs, their residual virulence and safety concerns regarding dosage remain significant (Urbano and Ferreira, Reference Urbano and Ferreira2022).

A third strategy for developing live attenuated ASFV vaccines involves the use of gene-editing techniques to change the virus genome and obtain deletion mutant strains. The quality of the gene-edited vaccine is controllable, and this approach can be used to distinguish between vaccine-immunized and wild-infected animals. Researchers from the European Union ASFV Reference Laboratory (Gallardo et al., Reference Gallardo, Sánchez, Pérez-Núñez, Nogal, de León, Carrascosa, Nieto, Soler, Arias and Revilla2018) in Spain used the ASFV wild-type strain NH/P68 as the backbone to construct multiple protein-deficient strains by using gene-editing techniques. After immunization, the vaccine candidates fully protected pigs against homologous challenge with L60, although they failed to protect against genotype II Arm07. The protection rate against this strain was thus unsatisfactory. The most promising candidate gene for an ASFV vaccine is a deletion mutant produced by homologous recombination or the CRISPR/Cas9 technique (Perez-Nunez et al., Reference Perez-Nunez, Sunwoo, Garcia-Belmonte, Kim, Vigara-Astillero, Riera, Kim, Jeong, Tark, Ko, You and Revilla2022; Rathakrishnan et al., Reference Rathakrishnan, Moffat, Reis and Dixon2020). Through these methods, reasonable deletions of virulence genes and interferon inhibitors have been attempted in several strains. The deleted virulence-related genes included 9GL (B119L), UK (DP96R), CD2v (EP402R), DP148R, and various members of the multigene family (MGF; Sang et al., Reference Sang, Miller, Lokhandwala, Sangewar, Waghela, Bishop and Mwangi2020). Along these lines, the deletion of different types of I interferon inhibitors can prevent the activation of the attenuated virus. The same deletion exhibits varying degrees of attenuation in different strains, and the duration of immunity remains a weakness of this potential ASF vaccine (Arias et al., Reference Arias, de la Torre, Dixon, Gallardo, Jori, Laddomada, Martins, Parkhouse, Revilla and Rodriguez2017). For example, the candidate vaccine HLJ/18-7GD, based on the highly virulent Chinese ASFV strain HLJ/18, was constructed by deleting gene fragments encoding 1–7 different proteins, including MGF505-1R, MGF505-2R, MGF505-3R, MGF360-12 L, MGF360-13 L, MGF360-14 L, and CD2v (EP402R) (Chen et al., Reference Chen, Zhao, He, Liu, Wang, Zhang, Li, Shan, Chen, Zhang, Wang, Wen, Wang, Guan, Liu and Bu2020). To examine whether these gene-deleted viruses were attenuated in pigs, specific pathogen-free (SPF) piglets were inoculated intramuscularly, and body temperature and survival rate were measured for 3 weeks. Except for HLJ/18-7GD, which was significantly weakened in pigs and exhibited a strong protective effect against the parents of the HLJ/18 strain and the genotype II Georgia07-like ASFV (Wang et al., Reference Wang, Zhang, Li, Zhang, Chen, Zhang, Sun, Zhu, Liu, He, Bu and Zhao2024), the other gene-deleted strains were lethal (Chen et al., Reference Chen, Zhao, He, Liu, Wang, Zhang, Li, Shan, Chen, Zhang, Wang, Wen, Wang, Guan, Liu and Bu2020). Recently, Borca and Tran reported a highly effective ASFV vaccine obtained by deleting the I177L gene via gene-editing techniques, named ASFV-G-DeltaI177L; this vaccine can induce effective protection against its parental virulent virus strain, Georgia 2007, as well as against a virulent Vietnamese field strain isolated in 2019. In addition, large-scale experiments to detect virus shedding and transmission have confirmed that even under varying conditions, ASFV-G-∆I177L remains a safe live-attenuated vaccine (Borca et al., Reference Borca, Ramirez-Medina, Silva, Vuono, Rai, Pruitt, Holinka, Velazquez-Salinas, Zhu and Gladue2020; Tran et al., Reference Tran, Phuong, Huy, Thuy, Nguyen, Quang, Ngon, Rai, Gay, Gladue and Borca2022). Based on the ASFV-G-∆I177L vaccine, Borca et al. (Reference Borca, Ramirez-Medina, Espinoza, Rai, Spinard, Velazquez-Salinas, Valladares, Silva, Burton, Meyers, Clark, Wu, Gay and Gladue2024) recently developed an ASFV-G-ΔI177L/ΔEP402R mutant by deleting the EP402R gene, which encodes the viral CD2v protein responsible for mediating haemadsorption of swine erythrocytes. This deletion not only attenuates the virus but also enables differentiation between infected and vaccinated animals (DIVA capability), a critical feature for vaccine-based control strategies. Currently, a new AVAC ASF LIVE vaccine, produced from an attenuated genotype II ASF virus (ASFV) strain with the deletion of six MGF genes and cultured in a Diep’s macrophage (DMAC) cell line, has been officially licensed for use and commercialization in Vietnam (Diep et al., Reference Diep, Ngoc, Duc, Dang, Tiep, Quy, Tham and Doanh2025). It is reported that this vaccine has demonstrated safety and high efficacy in protecting pigs from genotype II ASFV infection. However, to determine whether this vaccine can protect animals from heterologous ASFV infection, further investigation is needed. Certain naturally immunized pigs can resist attacks from homologous strains, but most of these pigs cannot resist attacks from heterologous strains (O’Donnell et al., Reference O’Donnell, Holinka, Sanford, Krug, Carlson, Pacheco, Reese, Risatti, Gladue and Borca2016). In fact, researchers have made progress in cross-protection using live attenuated ASFV vaccines. In 2017, Monteagudo reported a new recombinant live attenuated ASFV vaccine, named BA71∆CD2 (genotype I-based), in which deletion of the viral CD2v (EP402R) gene greatly attenuated the virulent BA71 strain in vivo (Monteagudo et al., Reference Monteagudo, Lacasta, Lopez, Bosch, Collado, Pina-Pedrero, Correa-Fiz, Accensi, Navas, Vidal, Bustos, Rodriguez, Gallei, Nikolin, Salas and Rodriguez2017). Inoculation of pigs with this kind of vaccine provided full protection not only against lethal challenge with the parental BA71 but also against the heterologous E75 (both genotype I strains). Interestingly, 100% of the pigs immunized with BA71ΔCD2 also survived lethal challenge with Georgia 2007/1, the genotype II strain of ASFV currently circulating mainly in continental Europe, Asia, and Oceania. In 2020, Lopez extended this research using BA71∆CD2 as a tool for understanding ASFV cross-protection via the use of phylogenetically distant ASFV strains. They first observed that five out of six (83.3%) of the pigs immunized once with 10⁶ PFU of BA71∆CD2 survived the tick bite challenge using Ornithodoros sp. soft ticks naturally infected with the RSA/11/2017 strain (genotype XIX, clade D, while BA71, E75 and Georgia2007/01 belonged to the same clade C). Second, they found that homologous prime boosting with BA71∆CD2 improved the survival rate only to 50% after Ken06.Bus (genotype IX, clade A) challenge, and all the infected pigs exhibited mild clinical signs consistent with ASF. However, they also noted that cross-protection is a multifactorial phenomenon that does not depend solely on sequence similarity, because they found that 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived lethal challenge with Ken06.Bus, with almost no clinical signs of this disease (Lopez et al., Reference Lopez, van Heerden, Bosch-Camos, Accensi, Navas, Lopez-Monteagudo, Argilaguet, Gallardo, Pina-Pedrero, Salas, Salt and Rodriguez2020). Later, researchers further identified that the onset of cross-protective immunity is triggered by the LAV candidate BA71∆CD2 after 12 days, accompanied by humoral immunity with the presence of virus-specific IgG and cellular immunity with a cytotoxic response before the challenge (Marin-Moraleda et al., Reference Marin-Moraleda, Munoz-Basagoiti, Tort-Miro, Navas, Munoz, Vidal, Cobos, Martin-Mur, Meas, Motuzova, Chang, Gut, Accensi, Pina-Pedrero, Nunez, Esteve-Codina, Gavrilov, Rodriguez, Liu and Argilaguet2024). Recently, Sereda developed another attenuated ASFV seroimmunotype I vaccine, named Katanga-350. The attenuated vaccine was shown to protect 80% of pigs from a virulent strain of the same genotype and seroimmunotype (Lisbon-57). In contrast, for heterologous ASFV strain attacks, at least 50% of the surviving pigs received protection from subsequent intramuscular infection with a heterologous (genotype II, seroimmunotype VIII) virulent strain (Stavropol 08/01) (Sereda et al., Reference Sereda, Vlasov, Koltsova, Morgunov, Kudrjashov, Sindryakova, Kolbasova, Lyska, Koltsov, Zhivoderov, Pivova, Baluishev, Gogin and Kolbasov2022). Although progress has been made in attenuated ASFV vaccines, potential problems, such as reversion of virulence and a shortage of a universal ASFV vaccine that can match most prevalent ASFV mutant strains, must be considered.

Genetically engineered vaccines

Compared to traditional inactivated vaccines and LAVs, genetically engineered vaccines may elicit more effective immune responses and greater safety (Chandana et al., Reference Chandana, Nair, Chaturvedi, Abhishek, Pal, Charan, Balaji, Saini, Vasavi and Deepa2024; Gaudreault and Richt, Reference Gaudreault and Richt2019; Goatley et al., Reference Goatley, Reis, Portugal, Goldswain, Shimmon, Hargreaves, Ho, Montoya, Sánchez-Cordón, Taylor, Dixon and Netherton2020). Genetically engineered vaccines for ASF include subunit vaccines, live virus vector vaccines, and nucleic acid (DNA) vaccines.