Molecular Characterization of ASFV and Differential Diagnosis of Erysipelothrix in ASFV-Infected Pigs in Pig Production Regions in Cameroon

Figures

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  Figure 1. The B646L gene phylogenetic tree constructed using isolates from this study (), isolates from previous studies in Cameroon () and other isolates representative of genotypes from GenBank (). The evolutionary tree was inferred by the neighbour-joining algorithm method with 1000 bootstraps, evolutionary distance computed using the p-distance method and 85% cut-off.

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  Figure 2. The E183L gene phylogenetic tree constructed using the ASFV isolates from this study, isolates from previous studies in Cameroon and other isolates representative of genotypes from GenBank. The evolutionary tree was inferred by the neighbour-joining algorithm method with 1000 bootstraps, evolutionary distance computed using the p-distance method and 85% cut-off. The samples in this study in sub-group Ia are marked in (), those from previous studies in Cameroon in () and other genotypes from GenBank in ().

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  Figure 3. The CP204L gene phylogenetic tree was constructed using the ASFV isolates from this study and other isolates representative of genotypes from GenBank. The evolutionary tree was inferred by the neighbour-joining algorithm method with 1000 bootstraps, evolutionary distance computed using the p-distance method and 85% cut-off. The samples in this study in genotype are marked in (), isolates from this study in no cluster in () and other genotypes from GenBank in ().

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  Figure 4. A multiple sequence alignment of the nucleotide sequences of the intergenic region between the I73R and I329L gene from this study with other isolates from Sardinia, Cagliari, Vietnam, Malawi, Korea and central Europe showing the GGAATATATA repeats and other deletions between isolates with single, double and triple GGAATATATA repeats.

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  Figure 5. Multiple sequence alignment using Clustal W within the Bio-edit software of the different variants within this study showing the deletion between the 19 repeats variants at the top and the six repeat variants below. The evolution of unequal crossing over and DNA slippage might be useful to uncover the mystery in this variation.

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  Figure 6. Comparative analysis of the partial EP402R gene of isolates within this study with other isolates with known serotypes. The seven repeats were separated between repeats four and five in 94.60% of the isolates by the SPPKPC and RPPPKC amino acid sequences, while CMR/Douala_SL6/2020, CMR/Douala_SL14/2020 and CMR/Douala_SL4/2020 had the SPPK-------PC thus lacking PCRPPK in between. Furthermore, isolates with the six and five PPPKPC amino acid repeats were both separated between repeats three and three and repeats four and five, respectively, by SPPKPC and RPPPKC amino acid sequence. We compared the isolates with other isolates from the Democratic Republic of Congo, Angola, Mozambique, and France with a determined number of repeats and known serotypes and found the seven PPPKPC amino acid repeats of this study clustered in serotype IV along with the isolate from France, but the five and six repeats did not cluster in any of the known serogroups.

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  Figure 7. Agarose gel of single 716 bp fragment amplified by PCR for Erysipelothrix differential diagnosis. PCR was performed using primer sets ERY-1F and ERY-2R along with MO101 and ERS-1R in a multiplex reaction. A single 716 bp fragment was amplified for all samples except for CMR/Douala_SL1/2020 and CMR/Buea_SL17/2020 with either a smear or no clear band, respectively.