Duck hepatitis A virus type 3 (DHAV-3), an avian-specific pathogen primarily impacting ducklings, poses a significant threat to the duck farming industry by causing high mortality rates. A DHAV-3 strain SH52 was isolated from the diseased ducks and phylogenetic analysis of the whole genome revealed that the DHAV-3 SH52 belongs to the prevalent strains in China. The DHAV-3 SH52 strain replicates at high levels in various organs of 8-day-old specific pathogen-free (SPF) shelducks, causing pathological damage and leading to high lethality in 8-day-olds following intramuscular infection. The findings of this study provide an ideal animal challenge model for evaluating the efficacy of DHAV-3 vaccines in the future.

In 2014, Duck Adenovirus type 3 (DAdV-3) emerged in Muscovy ducks and has since spread rapidly across China, causing significant economic losses to the duck industry. Given this situation, the development of reliable diagnostic tools is crucial for effective disease control. In this study, a neutralizing monoclonal antibody (mAb) 2F12 specific to DAdV-3 was generated, which showed a blocking rate of over 70% and a neutralization titer of up to 1:794. A blocking enzyme-linked immunosorbent assay (b-ELISA) was further developed based on mAb 2F12 to efficiently detect neutralizing antibodies against DAdV-3. The cut-off values of percent inhibition (PI) were set based on testing 84 negative duck serum samples, with a value below 16.79% (mean ($\overline{X}$) + 2 standard deviations (SD)) for negative sera and over 21.62% ($\overline{X}$ + 3SD) for positive sera. The b-ELISA exhibited a high specificity, reacting exclusively with DAdV-3 positive serum and showing no cross-reactivity with other representative positive sera tested. Additionally, the b-ELISA showed significantly higher sensitivity than the serum neutralization test (SNT), detecting antibodies 16-fold greater than the endpoint dilution of the SNT. The established b-ELISA, validated with 90 field serum samples from six duck farms, was well-suited for clinical detection of DAdV-3 antibodies and for monitoring post-vaccination antibody levels, representing a significant advancement in DAdV-3 detection and prevention.

A duck adenovirus type 3 strain, SD2019, was isolated from sick Muscovy ducks in our laboratory in 2019. To study the biological properties of the virus, an infectious clone of the SD2019 strain was successfully established. The plasmid containing the whole genome of DAdV-3 was digested with Pac I and the linearized DNAs were electortransfected into LMH cells; the cells showed cytopathic effects (CPEs) at 96 h post transfection and the rescued virus (rSD2019) was identified by PCR and indirect immunofluorescence assays (IFAs). The biological characteristics of strain rSD2019 were studied in vitro and in vivo and the results show that rSD2019 grew to similar titers as compared with the wild-type SD2019 strain in LMH cells, as well as showing similar replication and virulence in Muscovy ducks. The establishment of a reliable reverse genetics system for DAdV-3 provides a foundation for future studies of DAdV-3.

A duck adenovirus type 3 strain SD2019 was isolated from sick Muscovy ducks in our laboratory in 2019. To study the biological properties of the virus, an infectious clone of the SD2019 strain was successfully established. The plasmid containing whole genome of DAdV-3 was digested with Pac I and the linearized DNAs were electortransfected into LMH cells, the cells showed cytopathic effects (CPEs) at 96 hours post transfection and the rescuded virus (rSD2019) was identified by PCR and indirect immunofluorescence assays (IFA). The biological characteristics of strain rSD2019 were studied in vitro and in vivo and the results showed that the rSD2019 grew to similar titers as compared with the wild-type SD2019 strain in LMH cells, and showed similar replication and virulence in Muscovy ducks. The establishment of a reliable reverse genetics system for DAdV-3 provides a foundation for future studies of DAdV-3.

Duck enteritis virus (DEV), an epornitic pathogen, causes substantial economic losses in the commercial duck industry and poses persistent risks to wild and migratory waterfowl populations. However, due to the large genomic capacity of the DEV, the understanding of the virulence-associated genes of DEV is still limited. In previous studies, we developed an attenuated strain E74 by serial passage of a virulent strain E1 on primary chicken embryo fibroblasts (CEFs). The bird experiment showed that the mortality rate of E1 on ducks reached 100%, and high-titered viruses were detected in all tested tissue samples. In contrast, the E74 virus has lost its pathogenicity in ducks and can only be detected at a relatively low viral load in the spleen. Furthermore, the E74 stimulated a significant increase in antibodies in the ducks at 7 days post-inoculation. To further investigate the molecular basis of the attenuation of DEV in ducks, the complete genomes of E74 and E1 were sequenced and analyzed. Compared with E1, E74 had a 5152 bp deletion in the UL region, which resulted in the lack of the hypothetical protein, LORF5, UL55 and LORF4 genes. To test the influence of the deletion on the viral pathogenicity, a rescued virus rE1-Δ5152 with the 5152 bp deletion in the UL region was generated on the E1 backbone. Animal experiments showed that the lethality of rE1-Δ5152 in ducks had disappeared. Those findings suggest that the hypothetical protein, LORF5, UL55, and LORF4 genes of DEV are associated with virus virulence, and the flexibility of this region provided excellent insertion sites for exogenous genes when DEV is used as a recombinant vaccine vector.