A Guide to Molecular Characterization of Genotype II African Swine Fever Virus: Essential and Alternative Genome Markers

Abstract

African swine fever is a contagious viral disease that has been spreading through Europe and Asia since its initial report from Georgia in 2007. Due to the large genome size of the causative agent, the African swine fever virus (ASFV), the molecular epidemiology, and virus evolution are analyzed by employing different markers. Most of these markers originate from single nucleotide polymorphisms or disparities in the copy number of tandem repeat sequences observed during the comparisons of full genome sequences produced from ASFVs isolated during different outbreaks. Therefore, consistent complete genome sequencing and comparative analysis of the sequence data are important to add innovative genomic markers that contribute to the delineation of ASFV phylogeny and molecular epidemiology during active circulation in the field. In this study, the molecular markers currently employed to assess the genotype II ASFVs circulating in Europe and Asia have been outlined. The application of each of these markers to differentiate between ASFVs from related outbreaks is described to implement a guideline to their suitability for analyzing new outbreaks. These markers do not signify the complete repertoire of genomic differences between ASFVs, but will be beneficial when analyzing the first outbreaks in a new region or a large number of samples. Furthermore, new markers must be determined via complete genome sequence analyses for enabling in-depth insights into the molecular epidemiology of ASFV.

Keywords: African swine fever; differentition; gene; marker; phylogeny.

Figure 1

Phylogenetic tree based on partial sequence of C-terminal region of B646L gene, representing all 24 genotypes of ASFV, using reference isolates from GenBank.

Figure 2

Nucleotide sequence alignment of the partial CVR gene. The six different groups identified in Europe and the RF are expressed in different colors, whilst the four variants unique to China are expressed in violet color.

Figure 3

Amino acid sequence alignment of the tetrameric tandem repeat sequences (TRSs) of the central variable region (CVR) of the B602L gene.

Figure 4

Nucleotide alignment of ASFV samples based on the sequences of the intergenic region (IGR) between I73R and I329L. Each arrow denotes one TRS; yellow arrows indicate TRSs identical in quantity to the reference isolate Georgia 2007/1; blue arrows represent additional TRS.

Figure 5

Nucleotide alignment of ASFV samples based on the partial sequence of the K145R gene and depicting only three groups. Blue color—group K145R-I, which is similar to Georgia 2007/1; gray color—group K145R-II isolates with one SNP (C-to-A transversion at position 434); orange color—group K145R-III isolates with two simultaneous SNPs (C > T at position 291 and C > A at position 434).

Figure 6

Nucleotide alignment of ASFV samples based on the partial sequence of the O174L gene, demonstrating the two groups. Group O174L-I is identical to Georgia 2007/1, whilst group O174L-II contains two 14nt TRS.

Figure 7

Nucleotide alignment of ASFV samples using the intragenic regions between ORFs A179L and A137R genes. Sample ASF_Hanoi_Vietnmam_2019 [MT166692] has only one copy of the 11 bp TRS sequence, compared with the two copies in Georgia 2007/1.

Figure 8

Nucleotide alignment of ASFV isolates based on the analysis of the partial sequence of (A) MGF-505-5R and (B) MGF-110-7L, showing the SNP in these regions among genotype II isolates.

Figure 9

Nucleotide sequence alignment of ASFV samples using the partial ORF I267L gene. Samples from Poland, China, and the east of Russia contain an “A,” whilst the reference sequence Georgia 2007/1 and the western region of the RF contain a “T”.

Figure 10

Nucleotide sequence alignment of ASFV samples using the partial ORFs MGF-360-10L (A) and MGF-505-9R (B) genes. A. The T-to-C substitution in the MGF-360-10L gene at position 677 from the starting codon of the gene. B. An A-to-G substitution in the MGF-505-9R gene at position 967 from the starting codon of the gene.

Figure 11

Nucleotide alignment of ASFV samples using the intragenic regions between ORFs C315R and C147L. Sample Georgia 2008/2 [MH910496] has two copies of the 13 bp TRS sequence, compared with the single copy in Georgia 2007/1.