УДК 619.5:6616-635.5 RESEARCH ADVANCES IN AFRICAN SWINE FEVER VIRUS (minireview)

African swine fever remains one of most economically threatened diseases that has been hurting to the swine industry in Ukraine since 2014 and in China since 2018. African swine fever is an acute, highly lethal infectious disease caused by African swine fever virus, which has occurred and spread in many countries around the world, causing a catastrophic blow to the swine industry in the affected countries. ASFV is characterized of large genome, encoding 150-200 proteins, including variety of immunoregulatory proteins, which can resist immunity. African swine fever virus mainly enters pigs through the respiratory and digestive tract. The target cells infected are mainly mononuclear-macrophages, and the receptor is still unclear. Research on the development of diagnostic techniques and tests related to African swine fever are continuing and their proper using is crucial. There are many studies on African swine fever virus vaccines, including inactivated vaccines, attenuated vaccines, subunit vaccines and genetic vaccines. But so far these vaccines have not been able to protect domestic pigs from African swine fever virus infection. The article mainly reviews the researches of ASF virus, epidemiology, pathogenesis, diagnostic techniques and attempts to vaccine`s develop, that provides theoretical basis for the prevention and control of ASF.

Because the ASFV virulent strain genome can encode a variety of proteins to interfere with the host's natural immune system, so as to inhibiting and evading the host's immune response, creating favorable conditions for its own proliferation and spread, there is no effective vaccine for ASF prevention and treatment (Perez-Nunez, D. et al., 2015). Therefore, the development of vaccines is of great significance in controlling the disease. In addition, pathogenicity and pathogenesis research as an important part of animal virus research is the theoretical basis for studying animal diseases. This paper closely follows the new development trend of ASF international epidemic situation, and summarizes the research situation of ASFV from pathogens, epidemiology, pathogenic mechanism, diagnostic technology and vaccine, and provides theoretical basis for the prevention and control of ASF.
Etiology. ASFV is the only member of the African swine fever virus family and the African swine fever virus genus, and has similarities with certain features of the iridescent virus family and the poxvirus family. ASFV is a single-molecular linear doublestranded DNA virus, encapsulated by a capsule, icosahedral symmetrical, with a diameter of only 175-215 nm. There is a hole in the capsid center of the virus particle, making its structure a special six hexagon prism (Dixon L.K., Abrams C.C., Chapman D.G., et all, 2008). The ASFV genome is 170-190 kb in length and has 151 open reading frame ORFs encoding a total of about 150-200 proteins. The mature particles of the virus contain more than 50 major proteins, which play an important role in the infection process (Jia, N., Ou, Y., Pejsak, Z., Zhang, Y. & Zhang, J., 2017). Among them, P72 accounts for 1/3 of the total protein volume of virions, has a conserved protein sequence, and has good antigenicity. It can produce high titer anti-P72 antibody after infection, and is usually used for serological diagnosis of African swine fever. According to the nucleic acid sequence of the C terminal of the P72 gene, African swine fever can be divided into 24 genotypes (Achenbach, J. E. et al., 2017). The genome of the African swine fever virus is susceptible to variability, and the genetic processes are diverse, making it difficult to induce the production of neutralizing antibodies, so the serotype is not yet classified. By using restriction endonuclease digestion analysis, it was found that ASFV from America and Europe is the same genotype, while the isolated African strain has multiple genotypes, indicating that there were significant genotype differences between strains from different regions. The first strain to determine the full sequence of the genome is a non-toxic Spanish strain of BA71V, often used as the subject of laboratory study (Rodríguez, J. M., Moreno, L. T., Alejo, A., Lacasta, A., Rodríguez, F. and Salas M.L., 2015). At present, the whole ASFV gene sequences of 11 strains have been determined. One of them is virus (ASFV/Kyiv/2016/131) isolated from the spleen of a domestic pig in Ukraine with a lethal case of African swine fever. Using only long-read Nanopore sequences, we assembled a fulllength genome of 191,911 base pairs in a single contig. (Kovalenko G., Ducluzeau, A-L, Ishchenko, L., Sushko, M et al., 2019) ASFV is highly resistant to the external environment and can withstand a fairly wide pH (pH 4~13). It lasts for half a year in blood, feces and tissues. It lasts for up to 3 months in infected raw or undercooked pork products and can survive for several years in frozen meat (Junwei Wang, Zhiliang Wang, 2010). The virus can be inactivated at 60℃ for 20 minutes and can be inactivated a lipid solvent and a partial disinfectant (p-phenyl phenol disinfectant).
Epidemiology. European wild boars, warthogs, jungle pigs, giant forest pigs, sick pigs, rehabilitated domestic pigs and African soft palate are long-term sources of ASFV infection (Jori, F.; Bastos, A.D., 2009). Blome, S.; Gabriel, C.; Beer, M., 2013). Once the disease is established as endemic in an area, animals that survive for over a month are able to recover from the infection and even remain sub-clinically infected. But the role of survivor animals in the maintenance of the disease is still unclear.
The whole blood, tissues, secretions and excretions of the affected pigs and dead pigs contain viruses. Studies have shown that oral administration of the virus (dosage of10 5 HAD50/mL) or nasal (dose of 10 2.9 HAD50/mL) can cause infection in pigs; the acute infection period does not exceed 7 to 13 days, and the latent infection only appear in Europe before. Some pigs infected with attenuated strains; wild boar infections lasted for 21 days (Guinat, C. et al., 2016).
Africa's Ornithodoros soft palate not only carries ASFV for a long time, but also transmits pathogens vertically to offspring (XiaoJun Yang, Ze Chen, Jingze Liu, 2008). The ability of the ASFV to survive within particular ecosystems is defined by the biology of its wild host populations and also the features of livestock production systems, which influence host and vector species densities and interrelationships (Costard, S.; Mur, L.; Lubroth, J.; Sanchez-Vizcaino, J.M.; Pfeiffer, D.U., 2013).
ASFV mainly has three methods of spreading: direct contact propagation, indirect contact propagation and vector tick transmission. Direct contact spread involves domestic pigs and wild boars. Whether it is a domestic pig or a wild boar, once it is in the same line as a healthy pig after illness, it will cause infection in the herd. Wild boar plays an important role in ASFV transmission. The body of the wild boar that died of the disease and the soil in which the corpse rots are important factors in the spread of ASFV (Jori, F.; Bastos, A.D., 2009). However, wild boars in different countries have played a different role in ASFV transmission. For example, when studying the ASFV epidemic in Sardinia, Italian scientists believed that wild boars did not play a big role in the spread of ASFV. They believed that as long as the swine epidemic is extinguished, the wild boar epidemic will be purified (Mur, L. et al., 2016).
Indirect contact transmission: one is to feed waste containing infectious meat; the other is through illegal trade channels to purchase infected pigs, contaminated litter or feces, swill, etc. Contaminated vehicles, equipment, and clothing may also cause ASFV transmission when environmental pollution is

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Серія «Ветеринарна медицина», випуск 4 (47), 2019 severe. The spread of ASFV may cause by vaccination and drug treatment, such as poor disinfection or replacement of contaminated needles. Fresh grass and seeds contaminated by wild boar excreta can also cause spread of infection. African soft palate infects ASFV mainly by sucking infected pigs blood. For some virus isolates with high-propagation rate, almost 100% of susceptible sputum can replicate and maintain high titers at 4 weeks after the blood is saturated, and the virus in the sputum can also be transmitted In the affected areas, other blood-sucking insects, such as mosquitoes and flies, can also mechanically spread ASFV. The flies can spread ASFV after inhaling the blood of infected pigs for 24 hours, and can carry ASFV with high blood titer for more than 48 h. Blood louse can also carry viruses. In 1921, Montgomery managed to infect white rats, guinea pigs, rabbits, cats, dogs, goats, sheep, cattle, horses, pigeons and other animals are all unsuccessful. However, in 1956, Velho reported that pigs were killed by ASFV after the 26th generation of the rabbits. This indicates that the virus is only susceptible to pigs and does not cause infection to other animals (Guinat, C. et al., 2016).
Some researchers consider that African swine fever has high morbidity in naïve pig populations and can result in very high mortality (Costard, S.; Mur, L.; Lubroth, J.; Sanchez-Vizcaino, J.M.; Pfeiffer, D.U., 2013). But other researchers concern that defining ASF as "a highly contagious disease" can be delusive because it leads to false expectations and then underestimation of the problem (Guberti, V., 2018).
Pathogenic mechanism. ASFV enters pigs mainly through the respiratory tract and digestive tract. After the virus infects the body, it first proliferates in the tonsil, and then invades the whole body with blood and lymphatic system, triggers viremia, and replicates in vascular endothelial cells and macrophages. Invasion of blood vessels and lymphatics vessels causes pathological changes such as serous exudation, hemorrhage, thrombosis and necrosis in the corresponding organ tissues. pigs that died of diseases often show symptoms such as systemic organ and organ hemorrhage, impaired immune system, and decreased lymphocytes ( The main target cell of ASFV infection are porcine mononuclear-macrophages. Therefore, mononuclearmacrophages are the first place to detect the virus, followed by dendritic cells, endothelial cells, megakaryocytes, platelets, neutrophils and hepatocytes, can also detect ASFV (Munoz-Moreno, R., Galindo Vaccine research. In view of the huge impact of ASFV on the pig industry, the national economy and food safety, vaccine development has become the subject of research by many scientists, including inactivated vaccines, attenuated vaccines, genetically engineered vaccines, subunit vaccines and nucleic acid vaccines (Rock D L., 2017). (Table 1.). 2

Subunit vaccine
The ASF subunit vaccine uses a baculovirus as an expression vector to express a protective antigen of an African swine fever virus with a neutralizing epitope in a prokaryotic or eukaryotic cell, and then binds the obtained protein or polypeptide to an antigen presenting cell in order to induce higher anti-ASFV neutralizing antibodies. There are many kinds of structural proteins encoded by ASFV. Three important antigenic proteins P72, P54 and P30 have been found to have protective effects. Antibodies that produce P72 and P54 prevent viral adsorption, and antibodies to P30 prevent viral endocytosis. Recombinant proteins expressed in P30, P72 and P54 only delay clinical symptoms and reduce viremia levels, providing only 50% protection against effective protection. ASFV has complex structural proteins and immune evasion mechanisms, and it is difficult to obtain good immunoprotective effects against neutralizing antibodies produced by the above three antigens.

Viral live vector vaccine and live attenuated vaccine
At present, the viral live vector vaccine is mainly expressed in the induction of immune response. Related studies have used rabies virus, poxvirus or adenovirus as vectors to express ASFV protective antigen gene in order to obtain better cellular immunity and cytotoxic T lymphocyte (CTL) responses. In order to obtain an ideal immune response, a "cocktail" type of mixed immunization was used, but these studies did not carry out a challenge protection test, and the protective effect needs to be further verified to ensure the feasibility of the vaccine development method.

Vaccine based on ASFV receptor and protective antigen
Receptors and key antigens that currently mediate ASFV invasion of cells are still unclear. The cell challenge protection test has found that CD163 is a receptor of ASFV, and the expression level of CD163 is positively correlated with the degree of ASFV infection, but transgenic pigs with CD163 gene knockout by CRISPR/Cas9 do not show effective resistance to ASFV. Prove that CD163 is not a receptor of ASFV. Studies have shown that p12 protein may be a key antigen that mediates viral invasion, but in ASFV infection cells experiment, the addition of excess p12 antibody did not effectively block viral binding and infection, suggesting that p12 is not the only antigen that mediates viral adsorption. Studies have shown that p32, p72 and p54 also play important roles in the process of virus adsorption, p72 and p54 promote the binding of viruses and macrophages, while p32 contributes to virus internalization. What are the receptors for ASFV and need to be further explored. 6

Vaccine for oral immunization
Oral immunization of wild boar with a non-hemadsorbing, attenuated ASF virus of genotype II isolated in Latvia in 2017 (Lv17/WB/Rie1) conferred 92% protection against challenge with a virulent ASF virus isolate (Arm07). This is the first report of a promising vaccine against ASF virus in wild boar by oral administration. Further studies should assess the safety of repeated administration and overdose, characterize long-term shedding and verify the genetic stability of the vaccine virus to confirm if Lv17/WB/Rie1 could be used for free-ranging wild boar in ASF control programs.
There were many attempts to develop the vaccine that can provide protection against ASF virus, but protection was not 100%. The vaccine development and production has failed, mainly because ASFV has a complete anti-host vaccine response mechanism. The ASFV genome encodes a variety of proteins that interfere with the host's natural immune system, thereby inhibiting and evading the immune response, creating powerful conditions for its own proliferation and spread. In order to develop a vaccine that stimulates an effective anti-ASFV T-cell response Netherton, C. with collegs investigated which of the >150 viral proteins are recognized by the cellular immune response. The proteins capable of inducing ASFV-specific cellular and humoral immune responses in pigs were identified. Pools of viral vectors expressing these genes did not protect

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Серія «Ветеринарна медицина», випуск 4 (47), 2019 animals from severe disease, but did reduce viremia in a proportion of pigs following ASFV challenge (Netherton, C., et al., 2019). Diagnosis. The symptoms and lesions of African swine fever and classical swine fever are very similar, and the mortality rate of the disease is close to 100%, and the economic loss is huge, so there needs to be a rapid and effective laboratory differential diagnosis. At present, there are mainly the following main detection methods: First, PCR and real-time quantitative PCR detection methods for the conserved region of P72 gene can be used for rapid and effective detection of porcine spleen, blood, lymph The second is the ELISA method, which is applicable to the sandwich ELISA detection method for P72 protein in the spleen, blood, lymph nodes and other tissues. It also has whole virus or P72, P54 and other antigen coatings, and ELISA method for detecting serum antibodies. The third is the blood adsorption test. African swine fever virus has blood adsorption characteristics, and the red blood cells can be adsorbed on the infected macrophages to form a characteristic garland(Yunhao L., et al., 2014). The fourth is the direct immunofluorescence test, which is used to check pathogens in spleen, lymph nodes and other tissue sections. It has direct, rapid and effective characteristics, but only 40% of the subacute and chronic infections are detected because of the long course of disease. Some viruses have formed immune complexes, which are difficult to detect. The fifth the detection method is colloidal gold test paper, which has the advantages of rapidity, sensitivity and specificity, and is especially suitable for rapid clinical diagnosis. Zhang Xinyu et al. purified the ASFV p54 recombinant protein by using colloidal gold and spraying it on the fiberglass pad. The staphylococcal A protein (SPA) and anti-p54 polyclonal antibody were used as detection lines and quality control lines to prepare colloid gold immunochromatographic test strip for detecting of ASFV p54 antibody. The sensitivity of the test strip reached 200 ng/mL (Xinyu Z., Weiyong Z., Shanyuan Z., et al., 2014).
Outlook: ASF is a type of infectious disease with extremely complex pathogenesis and clinical symptoms. If we fail to detect and implement strict control measures at an early stage, they will spread rapidly and continue to spread, causing serious economic losses to society. The viability of ASF in ecosystems is determined by the relationship among the host, density of the vector, and habitat of the wildlife that affect ASF. The characteristics of livestock production system and the habitat of the soft ticks can also be affected.
There are three main characteristics of ASF epidemic: 1. In the spread of ASF, human factors account for a large proportion (such as hidden infections and sub-clinical infections when selling pigs); 2. low biosafety populations are more vulnerable to ASF invasion (the retail pigs are the main ASF attack group); 3. ASF is likely to spread to areas bordering epidemic areas. For these reasons, to better prevent and control the disease, it is necessary to have sufficient knowledge of its epidemiology in order to implement targeted measures.
Early vaccine research has focused on inactivated vaccines and attenuated vaccines. Numerous studies have shown that inactivated vaccines can induce antibodies in the body, but they have little protection. Although the attenuated vaccine has a certain protective effect on homologous strong virulence, due to the high variability of ASFV, its safety is very poor, which often causing diseases, leading to serious spread of pathogens. Spain and Portugal tried to use attenuated vaccines in the early days of ASF's introduction, but they all ended in failure. The use of attenuated vaccines led to large-scale and long-term epidemics, which seriously affected the process of disease control and elimination. In the past 20 years, molecular biology and immunology techniques have also been used to conduct a number of exploratory studies on vaccine and immune problems of ASF. ASFV in the process of adsorption and entry into cells, and P30, P54, P72 and other viral proteins are involved. Therefore, most of the ASF subunit vaccines and recombinant vaccines tend to select these genes, but subunits developed with baculovirus and other expression systems. Vaccines can only delay the onset of clinical symptoms and reduce the level of viremia to a certain extent, but it does not produce sufficient protection.