The increasing spread of African swine fever (ASF) in recent years and the presence of classical swine fever (CSF) subclinical forms in endemic countries suggests that the possibility of coinfection with ASF virus (ASFV) and CSF virus (CSFV) in pigs cannot be ruled out in areas where both diseases are prevalent. Thus, rapid and reliable diagnosis through molecular testing is essential for the timely implementation of control measures to prevent the spread of these devastating swine diseases. Here, we have coupled two of the most validated PCR assays for the detection of CSFV and ASFV in a single reaction tube. The combination of the two tests for the detection of two target nucleic acids did not affect the analytical sensitivity, and the duplex RT-qPCR assay was comparable with the standard molecular techniques. The detection limits for CSFV RNA and ASFV DNA were 0.12 TCID₅₀/reaction and 0.25 TCID₅₀/reaction, respectively. The test showed high repeatability and reproducibility, the coefficient of variation was below 2%, and excellent performance was demonstrated in clinical samples. The duplex assay shows great potential to become a robust diagnostic tool for the rapid and reliable detection and differentiation of CSFV and ASFV in areas where both viruses may be circulating.

Background/Objectives: Classical swine fever (CSF) continues to challenge global eradication efforts, particularly in endemic regions, where pregnant sows face heightened risks of vertical transmission following exposure to CSFV. Methods: This study evaluates the early protective efficacy of FlagT4G, a novel live attenuated DIVA-compatible vaccine. Pregnant sows were vaccinated at mid-gestation and challenged 14 days later with a highly virulent CSFV strain. Results: FlagT4G conferred complete clinical protection, preventing both maternal viremia and transplacental transmission. No CSFV RNA, specific antibodies, or IFN-α were detected in fetal samples from vaccinated animals. In contrast, unvaccinated sows exhibited clinical signs, high viral loads, and widespread fetal infection. Interestingly, early protection was observed even in the absence of strong humoral responses in some vaccinated sows, suggesting a potential role for innate or T-cell-mediated immunity in conferring rapid protection. Conclusions: The demonstrated efficacy of FlagT4G within two weeks of vaccination underscores its feasibility for integration into emergency vaccination programs. Its DIVA compatibility and ability to induce early fetal protection against highly virulent CSFV strains position it as a promising tool for CSF control and eradication strategies.

Classical swine fever (CSF) is a highly contagious transboundary animal disease that causes major economic losses to the global swine industry and remains notifiable to the World Organisation for Animal Health (WOAH). Handling and transport of infectious classical swine fever virus (CSFV)-positive samples require biosafety level 3 (BSL-3) containment, limiting diagnostic capacity and interlaboratory exchange in many regions. In this study, we evaluated the efficacy of PrimeStore^® Molecular Transport Medium (PS-MTM) for eliminating detectable CSFV infectivity while preserving viral ribonucleic acid (RNA) detectability for downstream molecular detection. Validation assays were performed using the CSFV Alfort/187 reference strain and clinical samples collected from pigs experimentally infected with the Catalonia01 and Margarita CSFV strains. Residual infectivity was assessed by virus isolation, whereas viral RNA detection and stability were evaluated using the WOAH-recommended real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR) assay. Following PS-MTM treatment, no residual infectivity was detected in either cell culture-derived virus stocks or clinical samples, including extensive replicate testing and three subsequent blind passages. RT-qPCR analyses confirmed preservation and stability of detectable CSFV RNA for at least 60 days under the storage conditions evaluated, including refrigerated storage and room temperature storage for Catalonia01-derived samples. Overall, these findings demonstrate that PS-MTM eliminates detectable CSFV infectivity while preserving viral RNA for molecular diagnosis. The implementation of PS-MTM may facilitate safer handling, transport, and interlaboratory exchange of CSFV-positive samples, contributing to improved biosafety, diagnostic harmonization, and global surveillance capacity for this transboundary animal disease.