Duck Tembusu virus (DTMUV) is a significant etiological agent responsible for egg production decline in laying ducks and retarded growth in ducklings, contributing to considerable economic losses in the poultry sector. Current research mainly focuses on the development of vaccines for the prevention of DTMUV. However, there is relatively little research on antiviral drugs against this virus. Curcumin has been reported to exert antiviral effects against multiple viruses. However, its efficacy against DTMUV remains unclear. This study aimed to investigate the antiviral activity of curcumin against DTMUV both in vitro and in vivo. Cytotoxicity in BHK-21 cells was assessed utilizing the Cell Counting Kit-8 (CCK-8) assay. The antiviral effects of curcumin were evaluated using viral titration, qRT-PCR, Western blotting, and indirect immunofluorescence. In addition, a DTMUV-infected duck model was used to assess the therapeutic potential of curcumin in vivo. The experimental results indicated that curcumin was non-toxic to BHK-21 cells at concentrations up to 30 μM. In vitro, curcumin significantly reduced DTMUV replication in a dose-dependent manner, as confirmed by decreased viral titers, RNA copy numbers, and E protein expression. Further analysis revealed that curcumin exerted its antiviral effect primarily at the post-adsorption stage of the viral life cycle. In vivo, curcumin treatment improved the survival rate of DTMUV-infected ducklings and alleviated virus-associated pathology. These findings demonstrate that curcumin effectively inhibits DTMUV infection and highlight its potential as a viable candidate for the prevention and treatment of DTMUV-related diseases.

Fowl adenovirus serotype 4 (FAdV-4) is the main causative agent of hydropericardium hepatitis syndrome (HHS), which has resulted in huge economic losses to the poultry industry in recent years. Hence, a rapid and simple visual detection method is needed for identification of FAdV-4. In this study, three multienzyme isothermal rapid amplification (MIRA) assays, basic MIRA, MIRA-qPCR and MIRA-LFD were developed for detection of FAdV-4. The amplification primers and reaction conditions were optimized, and the specificity and sensitivity of the assays were evaluated. The MIRA assays were specific for FAdV-4 with no cross-reaction with novel goose astrovirus, H9 subtype avian influenza virus, duck enteritis virus, Muscovy duck reovirus, or duck circovirus. The basic MIRA assay required only one primer pair and the reaction can be completed within 30 min at 36 °C. The MIRA-qPCR and MIRA-LFD assays were completed in 20 min with a minimum detection limit of 1 × 10¹ copies/μL and 1 × 10² copies/μL, respectively. The results of the MIRA-LFD assay can be observed directly with the naked eye, omitting the need for specialized instruments. The positive rate of three proposed MIRA assays were consistent with that of the conventional PCR assay. The MIRA assays are simple, rapid, and effective diagnostic tools for field detection of FAdV-4.

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.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to swine production, and its circulation after the African swine fever outbreak requires continued surveillance. This study investigated the temporal, regional, and genetic characteristics of PRRSV in Anhui Province from September 2019 to November 2024. Ten rounds of repeated cross-sectional surveillance were conducted at 147 slaughterhouses and 21 rendering plants. Tissue samples were collected by random, cluster, or risk-based sampling, pooled in groups of five, and tested by RT-qPCR. Representative positive samples with Ct values < 25 underwent ORF5 amplification, Sanger sequencing, and phylogenetic analysis. A total of 994 site visits yielded 18,733 tissue samples. No positive samples were detected in autumn 2020 or spring 2021, whereas PRRSV was detected again from autumn 2021 and subsequently fluctuated. The highest site-level positivity was 41.18% in autumn 2023, and the highest estimated individual-level positivity was 4.66% in spring 2023. Regional differences were statistically significant, with the highest site-level positivity in northern Anhui, and pooled-sample positivity was higher in rendering plants than in slaughterhouses. All 24 ORF5 sequences belonged to PRRSV-2 and mainly clustered with NADC30-like, NADC34-like, or MLV/classical strains. These findings demonstrate temporal fluctuations and lineage coexistence, supporting continued multisource surveillance and broader genomic and antigenic evaluation.

Goose astrovirus genotype 2 (GAstV-2) is an important pathogen associated with gosling gout, and rapid detection is useful for early diagnosis and field surveillance. In this study, a visual assay for GAstV-2 detection was developed by combining one-step reverse transcription multienzyme isothermal rapid amplification (MIRA) with the nucleic acid cleavage activity of Pyrococcus furiosus Argonaute (PfAgo). MIRA primers and specific guide DNAs were designed based on a conserved region of the GAstV-2 ORF1b gene, and the PfAgo reaction conditions were optimized. The optimal reaction contained 1.0 μM gDNA, 0.6 μM PfAgo, and 1.0 mM MnCl₂. Using recombinant pUC57-ORF1b plasmid DNA as the template, the lowest detectable plasmid concentration under the tested conditions was 1.0 × 10⁰ copies/μL. In the specificity assay, only GAstV-2 produced a positive signal, with no cross-reaction observed with GAstV-1, Tembusu virus, H9-subtype avian influenza virus, goose circovirus, fowl adenovirus serotype 4, or goose parvovirus. The assay was further tested with 23 clinical samples suspected of GAstV-2 infection. In a preliminary evaluation of 23 clinical samples, the RT-MIRA-PfAgo results were concordant with those obtained by conventional RT-PCR and RT-qPCR. Overall, the RT-MIRA-PfAgo assay provided sensitive and specific GAstV detection within a short time, without requiring programmed thermal cycling or an expensive real-time PCR instrument for routine endpoint detection. This method may be useful for GAstV-2 detection in basic laboratories and field settings.