First identified as a new circovirus in Hunan Province in China in 2019, porcine circovirus (PCV4) is now widely detected in other Chinese provinces and South Korea. In recent years, the virus has threatened pig health and operations in the pig industry. Hence, early PCV4 detection and regular surveillance are required to control the spread of infection and prevent collateral damage to the industry. Due to PCV4 being difficult to isolate in vitro, molecular detection methods, such as conventional PCR and real-time PCR, and serological assays are currently the main methods used for the detection of PCV4 infection. However, they are time-consuming, labor-intensive, and complex and require professional personnel. To facilitate rapid pen-side PCV4 diagnoses, we used clustered regularly interspaced short palindromic repeats (CRISPR) and Cas13a technology to develop a quick testing kit. Five recombinase-aided amplification (RPA) primer sets were designed based on the conserved PCV4-Cap gene nucleotide region, which were used to determine several key lateral flow strip (LFD) characteristics (sensitivity, specificity, and accuracy). The results showed that the RPA-Cas13a-LFD reaction could detect PCV4 within 1.5 h in genomic DNA harboring a minimum of a single copy. Furthermore, the assay showed good specificity and absence of cross-reactivity with PCV2, PCV3, or other porcine viruses. When we tested 15 clinical samples, a high accuracy was also recorded. Therefore, we successfully developed a detection assay that was simple, fast, accurate, and suitable for on-site PCV4 testing.

The high mortality rate of weaned piglets infected with porcine epidemic diarrhea virus (PEDV) poses a serious threat to the pig industry worldwide, demanding urgent research efforts related to developing effective antiviral drugs to prevent and treat PEDV infection. Small molecules can possibly prevent the spread of infection by targeting specific vital components of the pathogen’s genome. Main protease (Mpro, also named 3CL protease) plays essential roles in PEDV replication and has emerged as a promising target for the inhibition of PEDV. In this study, wogonin exhibited antiviral activity against a PEDV variant isolate, interacting with the PEDV particles and inhibiting the internalization, replication and release of PEDV. The molecular docking model indicated that wogonin was firmly embedded in the groove of the active pocket of Mpro. Furthermore, the interaction between wogonin and Mpro was validated in silico via microscale thermophoresis and surface plasmon resonance analyses. In addition, the results of a fluorescence resonance energy transfer (FRET) assay indicated that wogonin exerted an inhibitory effect on Mpro. These findings provide useful insights into the antiviral activities of wogonin, which could support future research into anti-PEDV drugs.`

Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV P72 gene, CSFV 5’UTR region, PRRSV ORF6, PCV2 cap gene, and PRV gB gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control.

The porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. However, there is no consensus on the primary receptor associated with the PEDV invasion of host cells. An increasing number of studies have reported that PEDV invading host cells may require collaboration between multiple receptors and to better understand the virus-host interaction during PEDV entry, surface plasmon resonance (SPR) assays are performed to investigate relevant host factors interacting with PEDV spike-1 protein (S1) in Vero and IPEC-J2 cell membranes. Subsequently, the rabbit anti-PEDV S1 polyclonal antibody is used as bait to recognize the complexes of IPEC-J2 membrane proteins with or without PEDV infection, followed by detection using liquid chromatography with tandem mass spectrometry (LC-MS-MS). Our results show that 13 and 10 proteins interacting between the S1 protein and plasma membrane protein of Vero or IPEC-J2 can be identified. More specifically, a total of 11 differentially expressed interacting proteins were identified in IPEC-J2 membrane proteins after PEDV infection, compared to the uninfected group. Furthermore, we found that the differentially interacting protein CCR4-NOT complex 2 (CNOT2), identified in PEDV S1 with plasma membrane proteins of Vero cells, is involved in viral infection. The results show that the knockout of CNOT2 significantly inhibits PEDV replication in vitro. These data provide novel insights into the entry mechanism of PEDV.

Conventional Escherichia coli (E. coli) detection methods are often time-consuming, while molecular diagnostics typically rely on expensive thermocycling equipment. To address these limitations, this study developed a rapid nucleic acid detection method for E. coli by integrating multienzyme isothermal rapid amplification (MIRA) with Pyrococcus furiosus Argonaute (PfAgo)-mediated targeted cleavage. The conserved housekeeping gene phoA was selected as the target, and specific MIRA primers and 5′-phosphorylated guide DNAs (gDNAs) were designed accordingly. After exponential amplification at 39 °C, the amplicons were specifically recognized by PfAgo at 95 °C, leading to molecular beacon cleavage and generation of a detectable FAM fluorescence signal. Among the tested guides, gDNA6 showed the highest cleavage efficiency. Optimal performance was achieved with 1 μM PfAgo, 0.5 μM gDNA, and 5 mM MnCl₂. The optimized MIRA-PfAgo assay demonstrated a limit of detection of 10⁰ copies/μL, comparable to qPCR, and exhibited high specificity with no cross-reactivity against common enteric pathogens. In 28 clinical and environmental samples, the assay results were fully consistent with those of qPCR. Overall, the MIRA-PfAgo platform provides a rapid, sensitive, and specific approach for E. coli detection, demonstrating strong potential to reduce reliance on precision thermal cyclers for resource-limited applications.