Genetic variability of African Swine Fever virus in Serbia

Afrička kuga svinja (AKS) je virusno hemoragično oboljenje domaćih i divljih svinja visokog stepena letaliteta, koje uzrokuje enormne ekonomske gubitke u industriji svinja. Uzročnik afričke kuge je istoimeni virus, jedini pripadnik familije Asfarviride i roda Asfivirus (Alonso i sar., 2018). Bolest je prvi put zabeležena u Srbiji 2019. godine u populaciji domaćih svinja, a od 2020. godine i u populaciji divljih svinja. Od tada do danas broj svinja je smanjen za 7% što je više od prosečnog pada broja svinja u Evropi od 4%. Genom virusa AKS je dvolančana linearna DNK dužine 170-190 kbp u zavisnosti od genotipa, podgenotipa i broja ponavljajućih segmenata i kodira sintezu 170 virusnih proteina (Blome i sar., 2020). Održavanje virusa u prirodi zavisi od faktora sredine, prisustva prijemčivih jedinki (divljih svinja) i otpornosti virusa. Jedan od ključnih uslova za održavanje i cirkulaciju virusa je i visoka prevalencija infekcije kod domaćih svinja (>5%) jer se na taj način omogućava prelivanje infekcije na divlje svinje i zatvaranje kruga prenošenja (Aguilar-Vega i sar., 2023). Letalitet kod divljih svinja inficiranih visoko virulentnim sojem iznosi 100%. Virus ostaje infektivan 3 meseca u leševima divljih svinja koje, ukoliko nisu uklonjene, dugotrajno predstavljaju izvor infekcije. Brzina širenja infekcije u populaciji divljih svinja u Evropi iznosi 4-17 km godišnje (Anette i sar., 2020). Širenje AKS na veće razdaljine uslovljeno je antropogenim faktorom. Čovek kontaktom sa zaraženim ili uginulim divljim svinjama, hranjenjem svinja kontaminiranim proizvodima animalnog porekla, transportom zaraženih domaćih svinja iz jednog područija u drugo predstavlja mehanički vektor za širenje virusa. Na afričkom kontinentu zabeležena su 24 genotipa virusa koja cirkulišu između populacija bradavičastih svinja, mekih krpelja iz roda Ornithodorus spp. i domaćih svinja. Van Afrike zabeleženi su samo genotip I i genotip II virusa AKS. Za trenutnu epizootiju odgovoran je genotip II virusa. Autori Gallardo i sar. (2023) su na osnovu analize genoma virusa AKS zabaležili 24 genogrupe u okviru genotipa II virusa na tlu Evrope. Na osnovu analize B646L i E183L gena opisan je jedan genotip virusa (genotip II). Daljom analizom B602L gena definisana su dva podgenotipa (CVR1 i 2) sa većim brojem varijacija koje zavise od broja tačkastih mutacija. Na osnovu analize intergenskog segmenta (I73L - I329L), mogu se opisati četiri podgenotipa (I-IV) (Gallardo i sar., 2023). Laboratorijska dijagnostika afričke kuge se vrši primenom molekularnih metoda. Cilj ovog rada je bila analiza cikulišućih sojeva virusa AKS u Srbiji. Trijažna testiranja su vršena real-time PCR testom. Dok je dalja analiza vršena primenom klasičnog PCR testa i Sanger sekvenciranja. Za detaljnu karakterizaciju lokalnih sojeva virusa AKS odabrano je 95 uzoraka poreklom od domacih i divljih svinja obolelih od AKS u periodu od 2019-2023 godine. Amplifikovana su i sekvencirana četiri segmenta virusnog genoma (B646L, E183L, B602L i intergenski region između I73L - I329L). Na osnovu analize ovih delova genoma utvrđeno je prisustvo više različitih sojeva virusa u Srbiji. Istovremena cirkulacija nekoliko sojeva može biti posledica spontanih mutacija ili reintrodukcije virusa iz drugih zaraženih područja. Utvrđivanje promena u genskom materijalu smatraju se veoma značajnim informacijama jer mogu ukazivati na adaptaciju virusa i modifikaciju virulentnosti. Praćenje promena u genomu virusa, porsedno daje uvid u strukturu virusnih proteina. Dugotrajna cirkulacija virusa u populaciji divljih svinja može dovesti do pojave srednje i niskovirulentnih sojeva što predstavlja izazov za eradikaciju bolesti. Kod domaćih svinja, srednje i niskovirulentni sojevi mogu izazvati atipične kliničke znakove i produženi tok bolesti što otežava postavljanje sumnje, odlaže dijagnostiku i povećava rizik od širenja bolesti. Potencijalne promene u konzerviranom delu genoma mogu otežati dijagnostiku virusa. Stoga su izolacija i genska karakterizacija virusa afričke kuge svinja od izuzetnog značaja za razumevanje epizootiologije ove bolesti. Poznavanje genskih karakteristika lokalnih sojeva virusa omogućava praćenje njihovog evolutivnog razvoja i identifikaciju novih sojeva. Na osnovu ovih podataka treba vršiti procenu efikasnosti preduzetih mera za suzbijanje i kontrolu bolesti. Usled nedostatka komercijalno dostupne vackcine, primena rigoroznih biosigurnostih mera je od najvećeg značaja za sprečavanje unosa uzročnika u populaciju domaćih svinja. Stroge kontrole na granicama su neophodne radi sprečavanja reintrodukcije virusa iz drugih zaraženih područija, kao i brza reakcija u aktivnim žarištima radi sprečavanja daljeg širenja bolesti i većih ekonomskih gubitaka.African Swine Fever (ASF) is a highly lethal viral hemorrhagic disease affecting domestic and wild pigs, causing significant economic losses in the swine industry. The causative agent of ASF is a virus of the Asfarviridae family and Asfivirus genus (Alonso et al., 2018). The disease was first recorded in Serbia in 2019 in domestic pig populations, and since 2020, it has also been found in wild pig populations. Since then, the number of pigs has decreased by 7%, which is higher than the average decline in pig numbers in Europe (4%). The genome of the ASF virus is a double-stranded linear DNA, ranging from 170-190 kbp in length, depending on the genotype, subgenotype, and number of repetitive segments, encoding the synthesis of 170 viral proteins (Blome et al., 2020). The maintenance of the virus in the environment depends on environmental factors, the presence of susceptible individuals (wild pigs), and the virus's ability to survive in that environment. A key condition for the maintenance and circulation of the virus is a high prevalence of infection in domestic pigs (>5%), as it enables the spill-over of infection to wild pigs, completing the transmission cycle (Aguilar-Vega et al., 2023). The case fatality rate in wild pigs infected with highly virulent strains is 100%. The virus remains infectious for 3 months in carcasses of wild pigs, which, if not removed, serve as a long-term source of infection. The spread of infection in the wild pig population in Europe is estimated at 4-17 km per year, with anthropogenic factors playing a significant role in long-distance transmission (Anette et al., 2020). Human contact with infected or dead wild pigs, feeding pigs with contaminated animal-derived products, and the transport of infected domestic pigs from one area to another act as mechanical vectors for virus spread. In Africa, 24 genotypes of the virus have been recorded, circulating among populations of bush pigs, soft ticks of the Ornithodorus spp. genus, and domestic pigs. Outside of Africa, only genotype I and genotype II of the ASF virus have been identified. The current epidemic is caused by the genotype II virus. Based on the genome analysis of the ASF virus Gallardo et al. (2023), identified 24 genogroups within the genotype II virus in Europe. From the analysis of the B646L and E183L genes, one virus strain (genotype II) was described. Further analysis of the B602L gene defined two subgenotypes (CVR1 and 2) with a higher number of variations depending on the number of point mutations. Based on the analysis of the intergenic segment (I73L - I329L), four subgenotypes (I-IV) can be described (Gallardo et al., 2023). Laboratory diagnostics of African Swine Fever are conducted using molecular methods. The aim of this study was to analyze circulating strains of ASF virus in Serbia. Screening tests were performed using real-time PCR, while further analysis was conducted using conventional PCR and Sanger sequencing. For a detailed characterization of local strains of ASF virus, 95 samples from diseased domestic and wild pigs with ASF between 2019 and 2023 were selected. Four segments of the viral genome (B646L, E183L, B602L, and the intergenic region between I73L - I329L) were amplified and sequenced. Based on the analysis of these genome segments, the presence of multiple different strains of the virus was identified in Serbia. Simultaneous circulation of several strains may result from spontaneous mutations or virus reintroduction from other infected areas. Determining changes in the genetic material is considered highly significant as they can indicate virus adaptation and modification of virulence. Monitoring changes in the virus genome provides insight into the structure of viral proteins. Prolonged circulation of the virus in the wild pig population can lead to the emergence of moderately and low-virulent strains, posing challenges for disease eradication. In domestic pigs, these strains can cause atypical clinical signs and prolonged disease courses, complicating suspicion, delaying diagnosis, and increasing the risk of disease spread. Potential changes in conserved regions of the genome can hinder virus diagnostics. Therefore, the isolation and genetic characterization of ASF virus strains are of utmost importance for understanding the epizootiology of this disease. Knowledge of the genetic characteristics of local virus strains allows for monitoring their evolutionary development and identification of new strains. Based on these data, an assessment of the effectiveness of control measures should be conducted. Due to the lack of commercially available vaccines, the implementation of rigorous biosecurity measures is of paramount importance to prevent the introduction of the pathogen into the domestic pig population. Strict controls at borders are necessary to prevent the reintroduction of the virus from other infected areas, along with swift responses in active outbreak areas to prevent further disease spread and mitigate significant economic losses.Zbornik radova i kratkih sadržaj