ASFV genotyping, serogrouping and CVR sequence analysis
Representative ASFV positive samples from the 4 selected LPMs were selected for molecular characterization targeting four regions of the virus. The regions were the C-terminal end of the B646L gene encoding the p72 protein, the full length of E183L gene encoding p54 protein, central variable region (CVR) within theB602L and CD2v protein encoding the EP402R gene (Bastos et al., 2003; Gallardo et al., 2009; Malogolovkin et al., 2015b). ASFV sequences were generated commercially by Macrogen Inc. (Netherlands Europe) and LGC Genomic (Germany). The raw sequence data were assembled using Staden Package (http://staden.sourceforge.net/) and multiple alignments were performed using the CLUSTALX algorithm implemented in MEGA X (Kumar et al., 2018). Sequences for four gene segments of ASFV were compared to available sequences in the GenBank using the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast) with default settings. MEGA X was used for phylogenic analysis to a Neighbor-joining tree inferred using the Maximum Composite Likelihood method for p72 protein with data resampled 1,000 times in the bootstrap method. For the p54 protein encoded in E183Lgene, a Minimum Evolution method tree was inferred using the Maximum Composite Likelihood method and data resampled 1,000 times in the bootstrap method (Kumar et al., 2018). The CD2v protein sequences were inferred using Neighbor-joining method and data resampled 1,000 times in the bootstrap method. Sequences of ASFV genotypes and serogroups were retrieved from the Genbank for the construction of the phylogenetic trees for both p72, p54 and CD2v protein genes. The tandem repeat sequences (TRS) of CVR (B602L gene) from this study were translated to protein sequences using ExPASy (https ://web.expas y.org/translate/) with default software settings. The translated amino acid tetramers were matched with previously reported codes (Achenbach et al., 2017; Nix et al., 2006). ASFV TRS from this study were compared with 21 TRS of CVR previously reported in Nigeria and other West African countries. Sequences were deposited into GenBank with the following accession numbers: OL692379- OL692391 (p72 gene), OL692397-OL692406 (p54), OL692410-OL692419 (B602L gene) and OL780773- OL780778 (CD2v gene).