AUTHOR=Gallardo Carmina , Casado Nadia , Soler Alejandro , Djadjovski Igor , Krivko Laura , Madueño Encarnación , Nieto Raquel , Perez Covadonga , Simon Alicia , Ivanova Emiliya , Donescu Daniel , Milicevik Vesna , Chondrokouki Eleni , Nurmoja Imbi , Frant Maciej , Feliziani Francesco , Václavek Petr , Pileviciene Simona , Marisa Arias 

TITLE=A multi gene-approach genotyping method identifies 24 genetic clusters within the genotype II-European African swine fever viruses circulating from 2007 to 2022

JOURNAL=Frontiers in Veterinary Science

VOLUME=10

YEAR=2023

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2023.1112850

DOI=10.3389/fvets.2023.1112850

ISSN=2297-1769

ABSTRACT=<sec><title>Introduction</title><p>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.</p></sec><sec><title>Materials and methods</title><p>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 <italic>I73R</italic> and <italic>I329L</italic> genes and the <italic>O174L</italic> and <italic>K145R</italic> genes. For further discrimination, two new PCRs were designed to amplify the IGR between the <italic>9R</italic> and <italic>10R</italic> genes of the multigene family 505 (MGF505) and the IGR between the <italic>I329L</italic> and <italic>I215L</italic> genes. The sequences obtained were compared with genotype II isolates from Europe and Asia.</p></sec><sec><title>Results</title><p>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 <italic>I329L</italic>-<italic>I215L</italic> 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 <italic>9R</italic>-<italic>10R</italic> genes of the multigene family 505 (MGF505) revealed eight different variants circulating.</p></sec><sec><title>Discussion</title><p>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.</p></sec>