Efficient Biosafe Inactivation of Classical Swine Fever Virus While Preserving Viral RNA for Molecular Diagnosis

Figures

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  Figure 1. Experimental workflow for the evaluation of CSFV inactivation using PS-MTM. Cell culture-derived CSFV Alfort/187 and clinical serum and nasal swab samples from Catalonia01-infected pigs were treated with PS-MTM and assessed for residual infectivity in PK-15 cells. PBS-treated samples were included as infectivity controls. Abbreviations: CSFV, classical swine fever virus; PS-MTM, PrimeStore® Molecular Transport Medium; PK-15, porcine kidney-15 cell line; PBS, phosphate-buffered saline; TCID50, 50% tissue culture infectious dose.

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  Figure 2. Workflow for CSFV RNA stability assessment in PS-MTM-treated clinical samples. Serum, nasal, buccal, and rectal swab samples from pigs experimentally infected with the CSFV Margarita strain were stored at 4 °C and analyzed by RT-qPCR at day 0, day 30, and day 60 after PS-MTM treatment. Serum and nasal swab samples from pigs experimentally infected with the CSFV Catalonia01 strain were aliquoted after PS-MTM treatment, stored at 4 °C and room temperature, and analyzed by RT-qPCR before treatment, at day 0, day 30, and day 60. The workflow was designed to assess the preservation and detectability of CSFV RNA during post-treatment storage. Abbreviations: RNA, ribonucleic acid; RT-qPCR, real-time reverse transcription quantitative polymerase chain reaction.

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  Figure 3. Assessment of residual CSFV infectivity following PS-MTM treatment. (A) Four independently treated preparations of the CSFV Alfort/187 reference strain were evaluated in PK-15 cells at the 10−2 dilution, together with the corresponding PBS-treated infectivity control. (B) Serum and nasal swab samples collected from five pigs experimentally infected with the CSFV Catalonia01 strain were independently treated with PS-MTM and evaluated under the same conditions. Viral replication was assessed using the peroxidase-linked assay after three blind passages. Numbers indicate the technical replicates evaluated. Abbreviations: S, serum; NS, nasal swab; CC, cell control; VC, virus control.

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  Figure 4. CSFV RNA detectability in clinical samples following PS-MTM treatment. (A) CSFV RNA detectability in serum and nasal swab samples collected from pigs experimentally infected with the CSFV Catalonia01 strain. Samples were analyzed by RT-qPCR before treatment, immediately after PS-MTM treatment (day 0), and after 30 and 60 days of storage at room temperature or 4 °C. (B) CSFV RNA detectability in serum, nasal swab, rectal swab, and buccal swab samples collected from pigs experimentally infected with the highly virulent CSFV Margarita strain. Samples were analyzed by RT-qPCR immediately after PS-MTM treatment (day 0) and after 30 and 60 days of storage at 4 °C. In both panels, the dotted horizontal line indicates the assay positivity threshold (Ct = 40). S, serum; NS, nasal swab; RS, rectal swab; BS, buccal swab; RT, room temperature.