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Pseudorabies (PR) is an acute infectious disease of pigs caused by pseudorabies virus (PRV), which has caused great economic losses to the pig industry worldwide. Reliable and timely diagnose is crucial for the surveillance, control and eradication of PR. Here, a real‐time fluorescent recombinase‐aided amplification (real‐time RAA) assay was established to detect PRV. Primers and probes were designed based on the conserved regions of the PRV gE gene. The assay was specific for the detection of wild‐type PRV, showing no cross‐reactivity with other important porcine viruses (including PRV gE‐deleted vaccine strains). Analytical sensitivity of the assay was three 50% tissue culture infectious doses (TCID50) of PRV DNA per reaction with 95% reliability, which is comparable to that of a PRV‐specific real‐time PCR (qPCR) assay. In diagnosis of 206 clinical tissue samples, the diagnose accordance rate between the real‐time RAA assay and qPCR assay was 97.57% (201/206). Interestingly, the amplified products of real‐time RAA could be visualized under a portable blue light instrument, making it possible for the rapid detection of PRV in resource‐limited settings and on‐site screening. Therefore, our developed real‐time RAA assay is a diagnostic method for the rapid detection of PRV in the field. [ABSTRACT FROM AUTHOR]

Classical swine fever (CSF), which is caused by the CSF virus (CSFV), remains one of the most economically important diseases of the global swine industry. Rapid and reliable detection of CSFV is critical for controlling CSF. In this study, a novel fluorescent probe‐based real‐time reverse transcription recombinase‐aided amplification (rRT‐RAA) assay, targeting a highly conserved position within the 5′ non‐translated region (5′NTR) among all CSFV genotypes, was developed for the detection of CSFV. The assay is highly specific to CSFV and does not cross react with other important viruses. Sensitivity analysis revealed that the assay could detect two 50% tissue culture infectious dose (TCID50) of CSFV RNA per reaction at 95% probability, which is comparable to that of a documentary reverse transcription quantitative PCR (RT‐qPCR) assay for CSFV. The rRT‐RAA assay exhibited good reproducibility, with intra‐ and inter‐assay coefficient of variation values of <8.0%. Of the 135 samples (including 102 clinical tissue samples and 33 different cell culture isolates of CSFV), 50 and 52 samples were tested positive for CSFV by rRT‐RAA and RT‐qPCR, respectively. The coincidence rate between the two assays was 98.5% (133/135). Further linear regression analysis showed a significant correlation between the rRT‐RAA and RT‐qPCR assays with an R2 value of 0.8682. Interestingly, the amplification products of the rRT‐RAA assay could be directly observed with naked eyes under a portable blue light imager, making it possible for an on‐site testing. Our results indicate that the rRT‐RAA assay is a robust diagnostic tool for the rapid detection of CSFV. [ABSTRACT FROM AUTHOR]

Importance: As one of the main etiologic agents of infectious diseases in pigs, pseudorabies virus (PRV) infections have caused enormous economic losses worldwide. EP0, one of the PRV early proteins (EP) plays a vital role in PRV infections, but the mechanisms are unclear., Objective: This study examined the function of EP0 to provide a direction for its in-depth analysis., Methods: In this study, the EP0-deleted PRV mutant was obtained, and Tandem Mass Tag-based proteomic analysis was used to screen the differentially expressed proteins (DEPs) quantitatively in EP0-deleted PRV- or wild-type PRV-infected porcine kidney 15 cells., Results: This study identified 7,391 DEPs, including 120 and 21 up-regulated and down-regulated DEPs, respectively. Western blot analysis confirmed the changes in the expression of the selected proteins, such as speckled protein 100. Comprehensive analysis revealed 141 DEPs involved in various biological processes and molecular functions, such as transcription regulator activity, biological regulation, and localization., Conclusions and Relevance: These results holistically outlined the functions of EP0 during a PRV infection and might provide a direction for more detailed function studies of EP0 and the stimulation of lytic PRV infections., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Korean Society of Veterinary Science.)