Avian influenza viruses (AIV) pose a significant persistent threat to the public health and safety. It is estimated that there have been over 100 outbreaks caused by various H7 subtypes of avian influenza viruses (AIV-H7) worldwide, resulting in over 33 million deaths of poultry. In this study, we developed a recombinase-aided amplification combined with a lateral flow dipstick assay for the detection of hemagglutinin (HA) genes to provide technical support for rapid clinical detection of AIV-H7. The results showed that the assay can complete the reaction within 30 min at a temperature of 39°C. Specificity tests demonstrated that there was no cross-reactivity with other common poultry pathogens, including Newcastle disease virus (NDV) and infections bronchitis virus (IBV). The detection limit of this assay was 1 × 10¹ copies/μL, while RT-qPCR method was 1 × 10¹ copies/μL, and RT-PCR was 1 × 10² copies/μL. The κ value of the RT-RAA-LFD and RT-PCR assay in 132 avian clinical samples was 0.9169 (p < 0.001). These results indicated that the developed RT-RAA-LFD assay had good specificity, sensitivity, stability and repeatability and may be used for rapid detection of AIV-H7 in clinical diagnosis.

In order to investigate the prevalence of duck Tembusu virus (DTMUV) in several regions of China, this study conducted an epidemiological survey on 2674 avian throat swab samples (including chickens, ducks, geese, and pigeons) collected from seven provincial-level administrative regions in China in 2024. Following RT-qPCR testing, 198 positive samples were identified, demonstrating an overall positivity rate of 7.40% (198/2674) across the seven provinces included in the study. Subsequent virus isolation using BHK-21 cells led to successful isolation in 17 cases. Additionally, genetic evolution analysis of the partial NS5 gene was carried out on these 17 isolates through RT-PCR amplification and sequencing. The data analysis indicated that Guangdong Province had the highest positive detection rate, reaching 22.40% (86/384), followed by Henan at 12.24% (47/384). Among infected hosts, geese were primarily affected by DTMUV, with a positivity rate of 40.76% (97/238). The prevailing subgroup of DTMUV in circulation in China is subgroup 3.2. Farmer’s markets, wholesale markets, slaughterhouses, and poultry farms all showed evidence of DTMUV presence, indicating widespread contamination across diverse locations. This study examines the distribution, genetics, and phylogenetic features of DTMUV in China, which will enhance our comprehension of the epidemiological landscape of DTMUV in China.

Infectious bursal disease virus (IBDV) is an important avian pathogen that damages the bursa of Fabricius and continues to threaten the poultry industry. Pyroptosis is an inflammatory form of programmed cell death, but its contribution to IBDV-induced cellular injury remains incompletely defined. Here, using DF-1 cells, we found that IBDV infection was associated with caspase-3 activation, cleavage of gasdermin E (GSDME), generation of the pore-forming N-terminal fragment (GSDME-N), increased lactate dehydrogenase release, plasma membrane damage, and enhanced inflammatory cytokine-associated readouts, including increased interleukin (IL)-1β secretion and elevated IL-18 messenger RNA (mRNA) expression. IBDV infection also induced mitochondrial dysfunction, as evidenced by loss of mitochondrial membrane potential (ΔΨm), increased reactive oxygen species production, and activation of caspase-9, supporting the involvement of a mitochondria-associated caspase-9/3 pathway. Pharmacological inhibition of caspase-9 or caspase-3 reduced pyroptosis-associated readouts, indicating that mitochondrial damage contributes to IBDV-induced lytic cell death under the tested conditions. Expression of viral protein (VP)2 or VP5 alone caused detectable cytotoxicity but did not robustly reproduce GSDME cleavage, suggesting that viral proteins may contribute to upstream cellular stress that is further amplified during infection. Together, these findings support that IBDV engages a mitochondria-associated caspase-9/3–GSDME axis to promote pyroptosis-like lytic cell death in DF-1 cells.

Group A rotavirus (RVA), as an important pathogen causing pigeon diarrhea, has caused substantial economic losses for the pigeon breeding industry in recent years in China. To better understand the prevalence and genetic diversity of RVAs among pigeon flocks in China, a fluorescence quantitative RT-PCR assay for detection of RVAs was developed in this study. The fluorescence quantitative RT-PCR assay showed satisfactory specificity, sensitivity, repeatability, and reproducibility, with a limit of detection (LOD) of 1 × 10² copies/μL for RVA. Then, 645 pigeon swab samples collected from live bird markets in 10 provinces of China from 2023 to 2024 were tested for RVA with the fluorescence quantitative RT-PCR assay established in this study to characterize the current epidemiological status of pigeon-origin RVAs (PiRVAs). The results showed that the PiRVA positivity rate detected by the RT-qPCR assay was 12.24% (79/645) among pigeon flocks in China. Guizhou Province had the highest positive rate of PiRVAs (42.86%) among the 10 provinces sampled. The VP7 and VP4 gene of nine representative positive pigeon RVA samples were amplified by specific primers, and the phylogenetic analysis of these samples revealed that all RVAs among pigeons belonged to the G18P[17] genotype, though with certain genetic variations. This study established a useful tool for diagnosing Pigeon RVAs in practice, and provided valuable information to better understand the epidemiology of Pigeon RVAs among pigeon flocks in China.

Chicken infectious anemia virus (CIAV) is a major immunosuppressive pathogen of poultry, causing aplastic anaemia, lymphoid atrophy, and severe haematopoietic dysfunction in young chicks, thereby posing a substantial threat to global poultry health and production. In this study, a novel highly pathogenic CIAV strain, designated CIAV-GDHY230813, was isolated from young Mahuang chickens in China and subjected to comprehensive molecular and pathogenic characterization. Whole-genome sequencing and phylogenetic analysis revealed that CIAV-GDHY230813 belongs to I-a branch. Notably, the VP1 protein harbored a glutamine residue at position 394, a molecular marker strongly associated with high virulence, together with multiple amino acid substitutions, insertions, and deletions across the coding regions. Pathogenicity experiments in specific-pathogen-free (SPF) chicks demonstrated that infection with CIAV-GDHY230813 resulted in pronounced growth retardation, severe damage to immune organs, and increased mortality. From 3 to 21 days post-infection (dpi), body weights of infected chicks were significantly lower than those of the control group ( p < 0.01). Marked thymic and bursal atrophy, splenomegaly, and significantly reduced haematocrit levels were observed, indicating severe anaemia. Furthermore, CIAV infection led to a marked reduction in antibody titres against Newcastle disease virus vaccination by 4-fold to 8-fold, reflecting substantial suppression of humoral immune responses. Quantitative analysis of viral distribution showed significantly elevated viral loads in blood, liver, thymus, spleen, and bursa of Fabricius at both 14 and 21 dpi ( p < 0.001), with peak levels detected at 21 dpi. Collectively, these findings demonstrate the strong replicative capacity and high pathogenic potential of CIAV-GDHY230813, providing valuable insights into the molecular epidemiology and pathogenic mechanisms of CIAV in China, supporting improved surveillance and control strategies, and laying a solid foundation for the development of effective vaccines against CIAV.

Both Chicken anemia virus (CAV) and Gyrovirus galga 1 (GyVg1) belong to the family Anelloviridae and genus Gyrovirus. They are non-enveloped, single-stranded circular DNA viruses that can infect chicken populations, leading to similar clinical symptoms such as growth retardation and immunosuppression, making clinical differentiation challenging. This study established a duplex TaqMan real-time PCR assay to simultaneously detect CAV and GyVg1. Specific primers and probes were developed to target the VP3 gene of CAV (labeled with VIC) and the VP1 gene of GyVg1 (labeled with FAM). The optimization process involved fine-tuning reaction conditions, such as primer/probe concentrations and annealing temperature, through a systematic matrix approach. The assay demonstrated remarkable linearity under optimized conditions (0.4 μmol/L primers, 0.2 μmol/L probes, and an annealing temperature of 52 °C), yielding high R² values of 0.9973 for CAV and 0.9981 for GyVg1. The method exhibited high specificity, showing no cross-reactivity with other common avian pathogens. The limits of detection were 1.0 × 10⁰ copies/μL for CAV and 1.0 × 10¹ copies/μL for GyVg1, making it 10-fold more sensitive than conventional PCR. Intra-assay and inter-assay coefficients of variation were below 1.5% and 2.5%, respectively, indicating excellent reproducibility. When tested on 411 clinical samples from Shandong Province, CAV showed a positivity rate of 48.66% (200/411), GyVg1 18.73% (77/411), and a co-detection rate of 17.52% (72/411). Moreover, 25 complete genome sequences of GyVg1 (2376 bp each) were acquired from positive samples. These sequences exhibited a nucleotide identity ranging from 93.6% to 99.3% among the isolates and 92.2% to 99.6% with reference strains. The phylogenetic analysis, which was based on whole-genome sequences, categorized the Shandong isolates into four clusters (I–IV). Notably, the strains were dispersed across these clusters and were intertwined with references from various regions and hosts. This observation suggests the absence of distinct geographic clustering and implies frequent cross-regional and cross-species transmission. In summary, this duplex real-time PCR assay offers a sensitive, specific, and dependable method for concurrently detecting and distinguishing CAV and GyVg1. The study’s findings reveal a significant co-infection rate and genetic variation in GyVg1, underscoring the importance of increased surveillance and deeper exploration of its pathogenicity and transmission patterns.