Pigeon Newcastle disease, caused by pigeon paramyxovirus type 1 (PPMV-1), is a significant infectious disease in pigeons that can result in substantial mortality and poses a severe threat to the pigeon industry. The rapid and accurate onsite diagnosis of pigeon disease is crucial for timely diagnosis and the implementation of effective prevention and control measures. In this study, we established a rapid detection method for PPMV-1 based on recombinase-aided amplification (RAA) and CRISPR/Cas12a. The RAA primers target the conserved regions of the L gene for preamplification in clinical nucleic acid samples, followed by CRISPR/Cas12a detection of the target gene. Visualization could be achieved by combination with a lateral flow dipstick (LFD). This method demonstrated high specificity, showing no cross-reactivity with non-PPMV-1 samples. The sensitivity of the method assessed by fluorescence analysis reached 10⁰ copies/µL, and when it was combined with an LFD, the sensitivity was 10³ copies/µL. The constructed RAA-CRISPR/Cas12a-LFD visual detection method was applied to clinical sample testing and was found to enable the rapid and accurate detection of swab samples and tissue specimens. Its sensitivity was consistent with the current gold standard, quantitative real-time PCR results. The RAA-CRISPR/Cas12a-LFD detection method we developed provides a novel approach for the rapid, simple, precise, and specific onsite diagnosis of pigeon Newcastle disease.

Avian infectious bronchitis virus (IBV), a major pathogen in the poultry industry, evolves rapidly via genetic mutations and recombination. While recombination between field and live vaccine strains is well-documented, its direct role in viral virulence remains unclear. In this study, four novel IBV field strains (SX/2407, SX/2408, SX/2409, SX/2410) were isolated from a single layer farm in Shanxi Province, China, and genetically characterized. Comprehensive genomic analysis revealed that SX/2409 is a natural recombinant, with SX/2408 (co-isolated) as the major parent and a 4/91-like vaccine lineage as the minor parent. To assess the functional impact of this recombination, pathogenicity was compared in chicks. Notably, SX/2409 exhibited markedly enhanced virulence, with a significantly higher mortality rate (60%) than its major parent SX/2408 (20%). The other isolates, SX/2407 and SX/2410, caused 50% and 0% mortality, respectively. Our findings provide compelling field evidence that recombination with a live vaccine strain can directly drives the evolution of a more virulent IBV phenotype. These results highlight the potential evolutionary risks of current vaccination strategies and emphasize the critical need for robust molecular surveillance and development of safer next-generation vaccines.

Wild birds and waterfowl serve as the natural reservoirs of avian influenza viruses (AIVs). When AIVs originating from wild birds cross species barriers to infect mammals or humans, they pose a significant threat to public health. The H12 subtype of AIVs primarily circulates in wild birds, with relatively few isolates reported worldwide, and the evolutionary and biological characteristics of H12 subtype AIVs remain largely unknown. In this study, we analyzed the spatiotemporal distribution of H12 subtype AIVs worldwide and conducted a comprehensive investigation into the evolutionary and biological characteristics of an H12N2 virus isolated from a whooper swan in Central China. Phylogenetic analysis revealed that the H12N2 isolate belongs to the Eurasian lineage, with its HA gene likely originating from a duck-derived H12N5 virus and its NA gene potentially derived from an H9N2 virus, indicating that it is a complex reassorted virus. Animal experiments in domestic ducks and chickens demonstrated that the virus replicates at low levels in the respiratory tract of poultry and exhibits moderate horizontal transmission in ducks. However, it is capable of efficient horizontal transmission in chickens. Mouse infection experiments revealed that the virus could be detected in the nasal turbinates and lungs of mice, indicating that the H12N2 virus can infect mice without prior adaptation. In vitro studies revealed that the virus replicates efficiently in MDCK cells, with significantly higher titers than those in DF1 cells. These findings, combined with the mouse infection results, suggest that the H12N2 virus poses a potential risk of mammalian infection. This study provides valuable insights regarding the characteristics of the H12N2 virus and highlights the importance of ongoing surveillance and risk assessment of AIVs originating from wild birds.

Pigeon Newcastle disease, caused by pigeon paramyxovirus type 1 (PPMV-1), is a significant infectious disease in pigeons that can result in substantial mortality and poses a severe threat to the pigeon industry. Rapid and accurate onsite diagnosis of pigeon disease is crucial for timely diagnosis and the implementation of effective prevention and control measures. In this study, we established a rapid detection method for PPMV-1 based on recombinase-aided amplification (RAA) and CRISPR/Cas12a. The RAA primers target the conserved regions of the L gene for preamplification in clinical nucleic acid samples, followed by CRISPR/Cas12a detection of the target gene. Visualization could be achieved by combination with a lateral flow dipstick (LFD). This method demonstrated high specificity, showing no cross-reactivity with non-PPMV-1 samples. The sensitivity of the method assessed by fluorescence analysis reached 100 copy/µL, and when it was combined with an LFD, the sensitivity was 103 copies/µL. The constructed RAA-CRISPR/Cas12a-LFD visual detection method was applied to clinical sample testing and was found to enable rapid and accurate detection of swab samples and tissue specimens. Its sensitivity was consistent with the current gold standard, quantitative real-time PCR results. The RAA-CRISPR/Cas12a-LFD detection method we developed provides a novel approach for the rapid, simple, precise, and specific onsite diagnosis of pigeon Newcastle disease.

Pigeon Newcastle disease (PND), caused by pigeon paramyxovirus type 1 (PPMV-1), is an acute, highly contagious disease that induces neurological symptoms and gastrointestinal disorders in pigeons, posing substantial economic risks to the global pigeon industry. The hemagglutinin-neuraminidase (HN) protein of PPMV-1 is a key surface glycoprotein mediating viral adsorption, invasion, and egress, and serves as a critical target for vaccine development and diagnostic reagent design. In this study, we aimed to generate a specific monoclonal antibody (mAb) against the HN protein of PPMV-1 and identify its recognized epitope. We codon-optimized the conserved 198–390 amino acid (aa) region of the HN protein from PPMV-1 strain, followed by prokaryotic expression and purification to serve as the immunogen. Through cell fusion monoclonal antibody screening, we successfully established a hybridoma cell line that secretes mAb 4H3. Western blot and immunofluorescence assay (IFA) confirmed that mAb 4H3 specifically reacts with PPMV-1 strains but exhibits no cross-reactivity with the commonly used NDV vaccine strain Lasota. Epitope mapping with a panel of truncated HN protein fragments identified the minimal B-cell epitope recognized by mAb 4H3 as ³¹⁰ DRVWF ³¹⁴ . Sequence homology analysis showed this epitope is highly conserved across PPMV-1 strains. Collectively, our results offer a valuable tool for developing PPMV-1-specific diagnostic assays, as well as for investigations into HN protein function and the development of epitope-based vaccines against PND.