Background: From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus. Results: The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. Conclusion: The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

H5N1 and H9N2 viruses are important causes of avian influenza in China. H5N1 is typically associated with severe to fatal disease in poultry, while H9N2 is usually associated with mild disease. Differences in viral virulence prompted us to investigate whether innate immune responses would be differentially regulated following infection by H5N1 and H9N2 viruses. To address this hypothesis, expression of a panel of innate immune-related genes including IFN-α, IFN-β, Mx1, OASL, ISG12, IFIT5, IRF7, USP18, SST, and KHSRP in immortal DF-1 cells following H5N1 and H9N2 infection was analyzed and compared by real-time quantitative RT-PCR. Cells infected by either virus overall exhibited a similar expression profile for four ISGs (Mx1, OASL, ISG12, and IFIT5), IFN-α, IFN-β, and SST gene. However, two immune-regulatory genes (IRF7 and KHSRP) were not responsive to highly pathogenic H5N1 infection but were strongly up-regulated in DF-1 cells infected with low pathogenic H9N2 infection. The subtype-dependent host response observed in this study offers new insights into the potential roles of IRF7 and KHSRP in control and modulation of the replication and virulence of different subtypes or strains of avian influenza A virus.

Avian reovirus (ARV) is one of the main causes of viral arthritis, tenosynovitis, malabsorption syndrome (MAS), runting-stunting syndrome, and immunodepression. In recent years, due to the emergence of new ARV strains, outbreaks of the disease have brought significant economic losses to chicken flocks. To determine the prevalence of ARV in China from 2010 to 2024, a total of 409 tissue samples from different breeding farms were collected from chickens presenting clinical signs of lameness and swollen joints in various flocks located in 18 provinces. As performed on these tissue samples, the ARV-specific reverse transcription-polymerase chain reaction (RT-PCR) assay indicated 111 ARV-positive samples with a positive rate of 27.14%. After viral isolation from the necropsied chicken samples, 69 ARV strains were isolated, and specific sigma C (σC) genes were amplified and sequenced. The sequence analysis of σC genes showed that these 69 isolates were grouped into six clusters, including 14 ARV isolates from cluster I (20.29%), 12 ARV isolates from cluster II (17.39%), 3 ARV isolates from cluster III (4.35%), 8 ARV isolates from cluster IV (11.59%), 3 ARV isolates from cluster V (4.35%), and 29 ARV isolates from cluster VI (42.03%). Except for cluster V, each of the other five clusters could be divided into two subclusters. Homology analysis showed that ARV isolates in clusters II–VI had only 50.3 to 60.8% homology with the commercial S1133 vaccine strain which is derived from cluster I. The ARVs in subcluster Ia had high homology with the S1133 vaccine strain (93.5–98.0%), while the ARVs in subcluster Ib had a low homology with the S1133 strain (73.4–76.4%). Further, the cluster VI viruses, the main epidemic genotype in China, had only 50.3–55.7% homology with the S1133 strain. The results of the pathogenicity test showed that the representative strains of the six different clusters all caused swelling of the footpads in SPF chickens, and the incidence rate was not significantly different. The present study will be helpful in the understanding the prevalence of ARV strains in China and revealed the genetic differences between the ARV isolates and the commercial vaccine strain.

Duck circovirus (DuCV) is a major pathogen in duck farming that causes immunosuppression and increases the susceptibility to secondary infections. This study focused on a Chinese DuCV strain, designated SDDC, which was isolated and characterized from Cherry Valley ducks in Shandong Province in 2022. The SDDC strain was successfully propagated in specific-pathogen-free (SPF) duck embryos and confirmed by transmission electron microscopy. Genomic analysis identified a complete genome of 1994 bp, with a critical amino acid mutation (I to T) at the 160th position in the Cap protein, potentially influencing viral tropism. Pathogenicity studies in 2-day-old SPF ducks revealed a multi-organ infection, with the spleen as the primary target, exhibiting the highest viral load and significant histopathological alterations. Analysis of multi-organ viral dynamics indicated a detectable viral presence across target organs as early as 3 dpi, followed by a progressive decline in viral load until day 7. A pronounced replication peak was observed on day 14, after which a gradual viral clearance occurred. This study provides critical insights into the genomics, pathogenicity, and tropism of DuCV, establishing a foundation for elucidating its infection mechanisms, and developing targeted control strategies to protect duck farming.