Development Real-Time PCR Assays to Genetically Differentiate Vaccinated Pigs From Infected Pigs With the Eurasian Strain of African Swine Fever Virus

Figures

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  Figure 1. Sequences of primers and probes used in the qPCR reactions as well as multiple parameters calculated during these developments. Hairpin and dimer values are expressed in standard Gibbs free energy (ΔG kcal/mole). Designs were carried out using the RealTime qPCR Assay tool. Probes were labeled at 5′ with FAM (Fluorescein amidites) or VIC (Victoria) dyes and at 3′ with MGB-NFQ (Minor groove binder-non fluorescent quencher).

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  Figure 2. Graphic representation of the genetic coverage of different qPCR tests. The figure shows a phylogenetic analysis reconstructed by neighbor-joining using full-length sequences of multiple ASFV strains which may be potentially detected by different qPCRs designed in this study. Next to each clade representing multiple genotypes are expressed the identity of different primers and probes included in each qPCR. Values of 100% indicate no differences between viral sequences and different oligonucleotides. A value of 95.65%, for one I177L real-time PCR primer, is due to the presence of one mismatch between the forward primer and sequences of different genotypes. Numbers along the branches represent the bootstrap support values.

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  Figure 3. Amplification efficiency and analytical sensitivity determinations. (A) Ten-fold dilutions of a standard plasmid representing variable amounts of all gene targets were used to calculate the amplification efficiency and analytical sensitivity of multiple qPCRs. (B) Ten-fold dilutions of a stock of ASFV-G with a known titer was used to establish parameters of analytical sensitivity in terms of the capability of different qPCRs to detect minimal amounts of infectious ASFV. Amount of infectious virus is expressed as HAD50/mL units. In all cases results represent the average value of six replicates.

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  Figure 4. Detection of ASFV-G in the presence of recombinant viruses. The sensitivity of different real-time PCRs was evaluated using mixes obtained from combined infections using constant concentrations of recombinant viruses and variable amount of ASFV-G (A) ASFV-ΔI177L, (B) ASFV-G-Δ9GL/ΔUK, and (C) ASFV-ΔMGF. This evaluation was carried out in both the original virus mixes and after being passed once in porcine macrophage. The detection of recombinant viruses was conducted using specific qPCRs to detect the gene encoding the markers M-cherry (ASFV-ΔI177L) and β-Gus (ASFV-G-Δ9GL/ΔUK and ASFV-ΔMGF).

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  Figure 5. Diagnostic sensitivity. of the different qPCRs assessed using a set of blood samples with different viral titers (expressed in HAD50/ml) collected from pigs experimentally infected with ASFV-G (A). Comparative average CT values among the different qPCRs in the detection of viremic blood samples obtained from pigs infected with ASFV-G (B).

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  Figure 6. Diagnostic specificity. The diagnostic specificity of different qPCRs was assessed using a set of 108 blood samples collected from naïve pigs. Results from different tests represent the detection of nonspecific reactions after 40 (p72 and MGF-360-12L) or 37 (I177L and UK) cycles of amplification.

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  Figure 7. Evaluation the capability of qPCRs to differentiate vaccinated pigs with (A) ASFV-ΔI177L, (B) ASFV-G-Δ9GL/ΔUK, and (C) ASFV-ΔMGF from those infected with ASFV-G. Blood samples collected from pigs at different time points of vaccination were tested before and after (green boxes) one passage of 72 h in swine macrophage cell cultures. M-cherry and β-Gus q PCRs were used for the detection of the recombinant viruses, while p72 assay was used as a marker for the presence of ASFV.