INTRODUCTION: African swine fever (ASF) is a contagious viral disease of pigs and wild boar that poses a major threat to the global swine industry. The genotype II African swine fever virus (ASFV) entered the European Union (EU) in 2014 and since then fourteen countries have been affected, Italy and North Macedonia being the last in 2022. While whole genome sequencing remains the gold standard for the identification of new genetic markers, sequencing of multiple loci with significant variations could be used as a rapid and cost-effective alternative to track outbreaks and study disease evolution in endemic areas. MATERIALS AND METHODS: To further our understanding of the epidemiology and spread of ASFV in Europe, 382 isolates collected during 2007 to 2022 were sequenced. The study was initially performed by sequencing the central variable region (CVR), the intergenic region (IGR) between the I73R and I329L genes and the O174L and K145R genes. For further discrimination, two new PCRs were designed to amplify the IGR between the 9R and 10R genes of the multigene family 505 (MGF505) and the IGR between the I329L and I215L genes. The sequences obtained were compared with genotype II isolates from Europe and Asia. RESULTS: The combination of the results obtained by sequencing these variable regions allowed to differentiate the European II-ASFV genotypes into 24 different groups. In addition, the SNP identified in the IGR I329L-I215L region, not previously described, grouped the viruses from North Macedonia that caused the 2022 outbreaks with viruses from Romania, Bulgaria, Serbia and Greece, differentiating from other genotype II isolates present in Europe and Asia. Furthermore, tandem repeat sequence (TRS) within the 9R-10R genes of the multigene family 505 (MGF505) revealed eight different variants circulating. DISCUSSION: These findings describe a new multi-gene approach sequencing method that can be used in routine genotyping to determine the origin of new introductions in ASF-free areas and track infection dynamics in endemic areas.

Bulgaria NRL National Diagnostic and Research Veterinary Medical Institute Sofia Bulgaria

Czech Republic NRL State Veterinary Institute Jihlava Jihlava Czechia

Estonian NRL National Centre for Laboratory Research and Risk Assessment Tartu Estonia

European Union Reference Laboratory for ASF Madrid Spain

Faculty of Veterinary Medicine University Ss Cyril and Methodius in Skopje Skopje North Macedonia

Greece NRL Greek Ministry of Rural Development and Food FMD Virological Rickettsial and Exotic Diseases Athens Greece

Italy NRL Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche Perugia Italy

Latvia NRL Laboratory of Microbiology and Pathology Institute of Food Safety Animal Health and Enviroment BIOR Riga Latvia

Lithuania NRL National Food and Veterinary Risk Assessment Institute Vilnius Lithuania

Poland NRL National Veterinary Research Institute Puławy Poland

Republic of Serbia NRL Institute of Veterinary Medicine of Serbia Belgrade Serbia

Romania NRL Institute for Diagnostic and Animal Health Bucharest Romania

Zobrazit více v PubMed

Alonso C, Borca M, Dixon L, Revilla Y, Rodriguez F, Escribano JM. ICTV report consortium ICTV virus taxonomy profile: Asfarviridae. J Gen Virol. (2018) 99:613–4. 10.1099/jgv.0.001049 PubMed DOI

Dixon LK, Chapman DA, Netherton CL, Upton C. African swine fever virus replication and genomics. Virus Res. (2013) 173:3–14. 10.1016/j.virusres.2012.10.020 PubMed DOI

Achenbach JE, Gallardo C, Nieto-Pelegrín E, Rivera-Arroyo B, Degefa-Negi T, Arias M, et al. . Identification of a new genotype of African swine fever virus in domestic pigs from Ethiopia. Transbound Emerg Dis. (2017) 64:1393–404. 10.1111/tbed.12511 PubMed DOI

Quembo CJ, Jori F, Vosloo W, Heath L. Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype. Transbound Emerg Dis. (2018) 65:420–31. 10.1111/tbed.12700 PubMed DOI PMC

Njau EP, Machuka EM, Cleaveland S, Shirima GM, Kusiluka LJ, Okoth EA, et al. . African swine fever virus (ASFV): biology, genomics and genotypes circulating in Sub-Saharan Africa. Viruses. (2021) 13:2285. 10.3390/v13112285 PubMed DOI PMC

Sun E, Huang L, Zhang X, Zhang J, Shen D, Zhang Z, et al. . Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection. Emerg Microbes Infect. (2021) 10:2183–93. 10.1080/22221751.2021.1999779 PubMed DOI PMC

Rowlands RJ, Michaud V, Heath L, Hutchings G, Oura C, Vosloo W, et al. . African swine fever virus isolate, Georgia, 2007. Emerg Infect Dis. (2008) 14:1870–4. 10.3201/eid1412.080591 PubMed DOI PMC

Gallardo C, Fernández-Pinero J, Pelayo V, Gazaev I, Markowska-Daniel I, Pridotkas G, et al. . Genetic variation among African swine fever genotype II viruses, eastern and central Europe. Emerg Infect Dis. (2014) 20:1544–7. 10.3201/eid2009.140554 PubMed DOI PMC

OIE (World Organisation for Animal Health). African Swine Fever (ASF)—Situation Report 6. (2022). Available online at: https://www.woah.org/app/uploads/2022/02/asf-report6.pdf (accessed November 21, 2022).

Ge S, Li J, Fan X, Liu F, Li L, Wang Q, et al. . Molecular characterization of African swine fever virus, China, 2018. Emerg Infect Dis. (2018) 24:2131–3. 10.3201/eid2411.181274 PubMed DOI PMC

Gonzales W, Moreno C, Duran U, Henao N, Bencosme M, Lora P, et al. . African swine fever in the Dominican Republic. Transbound Emerg Dis. (2021) 68:3018–9. 10.1111/tbed.14341 PubMed DOI

Lubisi BA, Bastos AD, Dwarka RM, Vosloo W. Molecular epidemiology of African swine fever in East Africa. Arch Virol. (2005) 150:2439–52. 10.1007/s00705-005-0602-1 PubMed DOI

Nix RJ, Gallardo C, Hutchings G, Blanco E, Dixon LK. Molecular epidemiology of African swine fever virus studied by analysis of four variable genome regions. Arch Virol. (2006) 151:2475–94. 10.1007/s00705-006-0794-z PubMed DOI

Vilem A, Nurmoja I, Niine T, Riit T, Nieto R, Viltrop A, et al. . Molecular characterization of African swine fever virus isolates in Estonia in 2014-2019. Pathogens. (2020) 9:582. 10.3390/pathogens9070582 PubMed DOI PMC

Mazur-Panasiuk N, Wozniakowski G. The unique genetic variation within the O174L gene of polish strains of African swine fever virus facilitates tracking virus origin. Arch Virol. (2019) 164:1667–72. 10.1007/s00705-019-04224-x PubMed DOI PMC

Mazur-Panasiuk N, Walczak M, Juszkiewicz M, Wozniakowski G. The spillover of African swine fever in Western Poland revealed its estimated origin on the basis of O174L, K145R, MGF 505-5R and IGR I73R/I329L genomic sequences. Viruses. (2020) 12:1094. 10.3390/v12101094 PubMed DOI PMC

Tran HTT, Truong AD, Dang AK, Ly DV, Nguyen CT, Chu NT, et al. . Circulation of two different variants of intergenic region (IGR) located between the I73R and I329L genes of African swine fever virus strains in Vietnam. Transbound Emerg Dis. (2021) 68:2693–5. 10.1111/tbed.13996 PubMed DOI

Nguyen VT, Cho KH, Mai NTA, Park JY, Trinh TBN, Jang MK, et al. . Multiple variants of African swine fever virus circulating in Vietnam. Arch Virol. (2022) 167:1137–40. 10.1007/s00705-022-05363-4 PubMed DOI

Shi K, Liu H, Yin Y, Si H, Long F, Feng S. Molecular characterization of African swine fever virus from 2019-2020 outbreaks in Guangxi Province, Southern China. Front Vet Sci. (2022) 9:912224. 10.3389/fvets.2022.912224 PubMed DOI PMC

Gallardo C, Elsukova A, Wozniakowski G, Nieto R, Soler A, Sánchez-Vizcaíno J-M, e al. Sequencing of the tandem repeat sequences (TRS) within the intergenic region between the multigene family 505 9R−10R genes: additional tool for subtyping genotype II African swine fever (ASF) isolates. In: Proceedings of the 11th International Congress for Veterinary Virology 12th Annual Meeting of EPIZONE, Vienna, Austria: (2018), p. 71.

Mazloum A, Igolkin AS, Vlasova NN, Romenskaya DV. African swine fever virus: use of genetic markers in analysis of its routes of spread. Vet Sci Today. (2019) 3–14. 10.29326/2304-196X-2019-3-30-3-8 DOI

Rodriguez JM, Salas ML, Viñuela E. Genes homologous to ubiquitin-conjugating proteins and eukaryotic transcription factor SII in African swine fever virus. Virology. (1992) 186:40–52. 10.1016/0042-6822(92)90059-X PubMed DOI

Fernández-Pinero J, Gallardo C, Elizalde M, Robles A, Gómez C, Bishop R, et al. . Molecular diagnosis of African Swine Fever by a new real-time PCR using universal probe library. Transbound Emerg Dis. (2013) 60:48–58. 10.1111/j.1865-1682.2012.01317.x PubMed DOI

World Organisation for Animal Health (WOAH): 2021 . African swine fever. In: Manual of Diagnostic tests and Vaccines for Terrestrial Animals, Vol. 2, Chapter 3.9.1. (2021). Available online at: https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.09.01_ASF.pdf (accessed November 20, 2022).

Gallardo C, Anchuelo R, Pelayo V, Poudevigne F, Leon T, Nzoussi J, et al. . African swine fever virus p72 genotype IX in domestic pigs, Congo, 2009. Emerg Infect Dis. (2011) 17:1556–8. 10.3201/eid1708.101877 PubMed DOI PMC

Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. (2021) 38:3022–7. 10.1093/molbev/msab120 PubMed DOI PMC

Forth JH, Calvelage S, Fischer M, Hellert J, Sehl-Ewert J, Roszyk H, et al. . African swine fever virus - variants on the rise. Emerg Microbes Infect. (2023) 12:2146537. 10.1080/22221751.2022.2146537 PubMed DOI PMC

Alkhamis MA, Gallardo C, Jurado C, Soler A, Arias M, Sánchez-Vizcaíno JM. Phylodynamics and evolutionary epidemiology of African swine fever p72-CVR genes in Eurasia and Africa. PLoS ONE. (2018) 13:e0192565. 10.1371/journal.pone.0192565 PubMed DOI PMC

Sidi M, Zerbo HL, Ouoba BL, Settypalli TBK, Bazimo G, Ouandaogo HS, et al. . Molecular characterization of African swine fever viruses from Burkina Faso, 2018. BMC Vet Res. (2022) 18:69. 10.1186/s12917-022-03166-y PubMed DOI PMC

Minoungou GL, Diop M, Dakouo M, Ouattara AK, Settypalli TBK, Lo MM, et al. . Molecular characterization of African Swine fever viruses in Burkina Faso, Mali, and Senegal 1989-2016: genetic diversity of ASFV in West Africa. Transbound Emerg Dis. (2021) 68:2842–52. 10.1111/tbed.14240 PubMed DOI

Hakizimana JN, Nyabongo L, Ntirandekura JB, Yona C, Ntakirutimana D, Kamana O, et al. . Genetic analysis of African swine fever virus from the 2018 outbreak in South-Eastern Burundi. Front Vet Sci. (2020) 7:578474. 10.3389/fvets.2020.578474 PubMed DOI PMC

Wade A, Achenbach JE, Gallardo C, Settypalli TBK, Souley A, Djonwe G, et al. . Genetic characterization of African swine fever virus in Cameroon, 2010-2018. J Microbiol. (2019) 57:316–24. 10.1007/s12275-019-8457-4 PubMed DOI

Gallardo C, Okoth E, Pelayo V, Anchuelo R, Martín E, Simón A, et al. . African swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site. J Gen Virol. (2011) 92(Pt 2):432–44. 10.1099/vir.0.025874-0 PubMed DOI

Gallardo C, Mwaengo DM, Macharia JM, Arias M, Taracha EA, Soler A, et al. . Enhanced discrimination of African swine fever virus isolates through nucleotide sequencing of the p54, p72, and pB602L (CVR) genes. Virus Genes. (2009) 38:85–95. 10.1007/s11262-008-0293-2 PubMed DOI

Mazloum A, Van Schalkwyk A, Chernyshev R, Shotin A, Korennoy FI, Igolkin A, et al. . Genetic characterization of the central variable region in African swine fever virus isolates in the Russian Federation from 2013 to 2017. Pathogens. (2022) 11:919. 10.3390/pathogens11080919 PubMed DOI PMC

Goller KV, Malogolovkin AS, Katorkin S, Kolbasov D, Titov I, Höper D, et al. . Tandem repeat insertion in African swine fever virus, Russia, 2012. Emerg Infect Dis. (2015) 21:731–2. 10.3201/eid2104.141792 PubMed DOI PMC

Kolbasov D, Titov I, Tsybanov S, Gogin A, Malogolovkin A. African swine fever virus, Siberia, Russia, 2017. Emerg Infect Dis. (2018) 24:796–8. 10.3201/eid2404.171238 PubMed DOI PMC

Kim SH, Lee SI, Jeong HG, Yoo J, Jeong H, Choi Y, et al. . Rapid emergence of African swine fever virus variants with different numbers of a tandem repeat sequence in South Korea. Transbound Emerg Dis. (2021) 68:1726–30. 10.1111/tbed.13867 PubMed DOI PMC

Mazloum A, van Schalkwyk A, Shotin A, Igolkin A, Shevchenko I, Gruzdev KN, et al. . Comparative analysis of full genome sequences of African swine fever virus isolates taken from wild boars in Russia in 2019. Pathogens. (2021) 10:521. 10.3390/pathogens10050521 PubMed DOI PMC

Andraud M, Bougeard S, Chesnoiu T, Rose N. Spatiotemporal clustering and random forest models to identify risk factors of African swine fever outbreak in Romania in 2018-2019. Sci Rep. (2021) 11:2098. 10.1038/s41598-021-81329-x PubMed DOI PMC