During a field study about porcine reproductive and respiratory syndrome virus (PRRSV) in Danish pig herds, it was discovered that the quantitative real-time RT-PCR (RT-qPCR) assay used in the study was unable to detect PRRSV-2 in two herds (Herds A and B) exhibiting clinical signs consistent with PRRS. Two additional herds (Herd C, identified through clinical suspicion, and Herd D, identified during the field study) showed reduced assay sensitivity. Sequence analysis of viruses from all four herds demonstrated that the reduced sensitivity was caused by mutations within the primer and probe binding sites. Full genome analysis of Danish PRRSV-2 sequences revealed the existence of two phylogenetically separate mutated variants with a high degree of identity to previously identified Danish variants, suggesting that they were not newly introduced but likely arose separately in Denmark.
Hantaan virus (HTNV) and Seoul virus (SEOV) are the primary causative agents of hemorrhagic fever with renal syndrome (HFRS), presenting a persistent challenge to global public health. Prompt and precise diagnostic tools are essential for clinical investigation and the containment of HFRS outbreaks. Leveraging the programmable precision of CRISPR/Cas12b system, which has significantly advanced the field of molecular diagnostics, we developed a rapid, high-sensitivity assay for the dual detection of HTNV and SEOV. Each virus was detected in a separate, target-specific single-tube reaction. The assay combines recombinase polymerase amplification (RPA) with CRISPR/Cas12b-mediated fluorescence detection and uses virus-specific sgRNAs and RPA primers targeting conserved genomic regions. Using recombinant plasmid standards, both targets were detected at 1 × 101 copies/test in three technical replicates; this is a preliminary analytical detection threshold and does not incorporate extraction losses or matrix-associated inhibition. No cross-reactivity was observed within the evaluated four-organism non-target panel. In a preliminary feasibility assessment, all nine RT-qPCR-positive rodent samples (seven HTNV and two SEOV) were positive by the RPA-CRISPR/Cas12b assay, indicating complete positive agreement in this all-positive sample set. Because no negative samples were included, negative agreement, diagnostic specificity, false-positive rate, and overall clinical diagnostic accuracy could not be assessed. These results support further development of the assay as a potential screening tool; larger studies using well-characterized positive and negative clinical or field specimens are required.
In Atlantic salmon hatcheries, Salmon gill poxvirus (SGPV) can cause parachute, high mortality that can be alleviated by immediate countermeasures. However, this is hampered by a lack of good rapid diagnostics for SGPV. In this study, we developed and evaluated two rapid non-invasive isothermal amplification assays, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), each coupled with a lateral flow device (LFD) for detection of SGPV in gill mucus or lysates from SGPV-infected salmon. Both assays demonstrated high analytical specificity and strong agreement with the reference real-time PCR assay when purified DNA was tested. The RPA-LFD assay achieved sensitivity comparable to real-time PCR but was consistently inhibited by crude gill mucus, limiting its suitability for direct field testing. In contrast, the LAMP-LFD assay maintained high sensitivity and reproducibility using crude gill mucus without prior nucleic acid extraction, detecting SGPV in 14 of 15 field samples with excellent agreement with real-time PCR. Although analytical sensitivity of LAMP was approximately fourfold lower than real-time PCR, this did not compromise SGPV detection in relevant samples and a final mucus concentration of approximately 4% was optimal for SGPV amplification. Both assays showed no cross-reactivity with common salmon gill pathogens, including Candidatus Branchiomonas cysticola, Desmozoon lepeophtherii, and Paramoeba perurans. The LAMP-LFD workflow provides visual results within 45 min, representing a viable, rapid, and non-invasive diagnostic platform for field surveillance. These findings demonstrate that LAMP-LFD is well suited for SGPV detection and has strong potential to improve early outbreak detection and enhance the management of SGPV in salmon aquaculture.
Porcine epidemic diarrhoea virus (PEDV) can cause fatal watery diarrhoea in newborn piglets, posing a serious threat to the global pig industry. The spike (S) protein is the main envelope antigen of PEDV and an ideal target for antigen detection. In this study, we immunised rabbits to prepare antibodies directed to the receptor binding domain (RBD) and the S2 domain and used the ExpiCHO system to express five previously produced nanobodies that specifically recognise the PEDV S protein. The best antibody pairing scheme was determined via checkerboard titration, and a double antibody sandwich ELISA (DAS-ELISA) was established, using mixed RBD and S2-specific rabbit pAbs as the capture antibody and HRP-labelled Nb9 nanobody as the detection antibody. The cut-off value was set at 0.250–0.280 based on 66 negative anal swab samples. This method showed no cross-reactivity with other diarrhoea viruses. The detection limit for live virus was 102.6 TCID50/mL; when the OD450 values for S protein ranged from 0.0625 μg/mL to 3 μg/mL, there was a good linear relationship with antigen concentration. Both intra- and inter-batch coefficients of variation were below 10%, showing good repeatability. Analysis of 166 clinical faecal samples alongside RT-PCR gave an overall concordance of 95.78% with a κ value of 0.915. ROC curve analysis showed a sensitivity of 90.20% and specificity of 92.65%. In summary, the nanobody DAS-ELISA method established in this study can detect PEDV antigens with high specificity and sensitivity, providing a new approach for on-site PEDV surveillance and quantitative vaccine antigen assessment.
The global resurgence of mpox, caused by monkeypox virus (MPXV), has highlighted the need for effective antiviral strategies, rapid diagnostics, vaccination, and integrated preparedness approaches. This review critically examines recent advances in MPXV biology, pathogenesis, transmission, clinical manifestations, prevention, diagnosis, treatment, and emerging nanomedicine-based strategies within a One Health framework. Particular emphasis is placed on nanoparticle-enabled vaccines, diagnostics, therapeutics, and wastewater surveillance, highlighting their potential to improve antigen delivery, diagnostic sensitivity, targeted drug delivery, and population-level monitoring. The review also compares conventional and nanoscale approaches and evaluates the current evidence supporting their efficacy and translational potential. Importantly, most nano-enabled MPXV interventions remain at the preclinical stage, and their reported performance cannot be directly compared because of differences in nanoparticle composition, target antigens, experimental models, assay conditions, and clinical validation strategies. Major challenges include long-term safety, pharmacokinetics, reproducibility, scalability, storage stability, regulatory approval, and the limited availability of well-designed clinical studies. Within the One Health framework, effective MPXV control also requires coordinated surveillance of human, animal, and environmental reservoirs, including standardized wastewater monitoring and cross-sectoral data sharing. Future research should therefore prioritize rigorous clinical validation, standardized evaluation protocols, diverse MPXV clade testing, long-term safety assessment, scalable manufacturing, and integrated human–animal–environmental surveillance.
