Infectious bursal disease virus (IBDV), a double-stranded RNA virus, causes immunosuppression and high mortality in 3–6-week-old chickens. Innate immune defense is a physical barrier to restrict viral replication. After viral infection, the host shows crucial defense responses, such as stimulation of antiviral effectors to restrict viral replication. Here, we conducted RNA-seq in avian cells infected by IBDV and identified TRIM25 as a host restriction factor. Specifically, TRIM25 deficiency dramatically increased viral yields, whereas overexpression of TRIM25 significantly inhibited IBDV replication. Immunoprecipitation assays indicated that TRIM25 only interacted with VP3 among all viral proteins, mediating its K27-linked polyubiquitination and subsequent proteasomal degradation. Moreover, the Lys854 residue of VP3 was identified as the key target site for the ubiquitination catalyzed by TRIM25. The ubiquitination site destroyed enhanced the replication ability of IBDV in vitro and in vivo. These findings demonstrated that TRIM25 inhibited IBDV replication by specifically ubiquitinating and degrading the structural protein VP3.

In recent years, hepatitis-hydropericardium syndrome (HHS), caused by novel fowl adenovirus 4 (FAdV-4), has caused serious economic losses to the poultry industry. Vaccines are important for preventing and controlling HHS. Current FAdV-4 vaccine research and development are mainly focuses on inactivated vaccines and relatively fewer live vaccines. We previously demonstrated that the hexon gene is the key gene responsible for the high pathogenicity of FAdV-4 and constructed a non-pathogenic chimeric virus rHN20 strain based on the emerging FAdV-4. In this study, the immunogenicity of artificially rescued rHN20 was evaluated in chickens using different routes and doses as a live vaccine. The live rHN20 vaccine induced high titers of neutralizing antibodies against FAdV-4 and fully protected the immunized chickens against a lethal dose of FAdV-4. Furthermore, immunized chickens showed no clinical symptoms or histopathological changes in the FAdV-4-targeted liver, and the viral load in the tissues of immunized chickens was significantly lower than that of chickens in the challenge control group. Collectively, the live rHN20 vaccine effectively protected our sample against FAdV-4 infection and can be considered a live vaccine candidate for preventing HHS in the poultry industry.

Inclusion body hepatitis (IBH), hydropericardium syndrome, and gizzard erosion associated with fowl adenovirus (FAdV) infection have caused notable economic losses worldwide. In 2020, severe IBH was observed in a layer chicken farm in Hebei Province, China. Liver samples were collected from layer chickens with severe IBH and virus isolation was performed in LMH cells. DNA sequence and bioinformatics analyses were conducted to determine the phylogenetic relationship and the pathogenicity assay was conducted in specific-pathogen-free (SPF) chickens. HeB20 strain was isolated and identified as FAdV-8b, and the complete genome was successfully sequenced (GenBank No. OK188966). Although widespread recombination in clinical strains has been reported within FAdVs, HeB20 showed some novel characteristics, and did not show any recombination, highlighting that recombinant and non-recombinant FAdV-8b coexist in the clinic poultry industry. Finally, pathogenicity animal model of HeB20 was developed and showed severe IBH and 10% mortality. Collectively, a new FAdV-8b strain (HeB20) was isolated and responsible for the severe IBH in layer chickens. Complete genome of HeB20 was sequenced and valuable for future epidemiological investigations. HeB20 was capable of inducing severe IBH and 10% mortality in SPF chickens; this animal model provides a powerful tool for the future vaccine development.

Bat-origin swine acute diarrhea syndrome coronavirus (SADS-CoV) causes diarrhea and death in piglets and also poses a cross-species transmission risk due to its broad host cell tropism. In the absence of commercial vaccines or therapeutics, identifying the host factors exploited by SADS-CoV may provide potential targets for antiviral therapies. Inhibition of glycogen synthase kinase 3 (GSK3) restricts diverse coronavirus replication by targeting the N protein and disrupting its phosphorylation. Here, we identified GSK3 as a crucial host factor for SADS-CoV RNA synthesis. Both GSK3 deficiency and treatment with a classical GSK3 inhibitor, lithium chloride (LiCl), strongly impaired SADS-CoV dsRNA synthesis. LiCl treatment did not alter electrophoretic mobility or expression of SADS-CoV N protein in N-transfected Huh7 cells but significantly reduced NSP12 protein levels. The absence of GSK3 led to the proteasomal degradation of NSP12 to reduce viral RNA synthesis. However, GSK3 interacted with ubiquitin-specific peptidase 27X (USP27X) and promoted the USP27X-mediated deubiquitination of NSP12, thereby enhancing NSP12 stabilization and facilitating viral RNA synthesis. LiCl not only protected neonatal mice from lethal SADS-CoV challenge but also exhibited a notable broad-spectrum antiviral effect against PEDV and TGEV. Our findings demonstrate that GSK3-mediated NSP12 stabilization is essential for SADS-CoV RNA synthesis, underscoring the potential of GSK3 as a broad-spectrum antiviral target for emerging and re-emerging swine coronaviruses. IMPORTANCE: Zoonotic transmission of coronaviruses continues to pose a significant threat to human populations. Swine acute diarrhea syndrome coronavirus (SADS-CoV) has been identified as a potentially high-risk emerging coronavirus pathogen that could adversely affect the global economy and human health. However, the replication mechanisms of SADS-CoV remain largely unexplored. Our study demonstrates that GSK3 stabilizes NSP12 by interacting with USP27X, thereby enhancing the synthesis of SADS-CoV dsRNA. LiCl, a GSK3 inhibitor, exhibits broad-spectrum antiviral activity against swine enteric alphacoronaviruses. Our work is distinguished for the first time by the GSK3–USP27X regulatory axis that controls the stability of NSP12 and indicates that GSK3 is a promising therapeutic target for the development of broad-spectrum antiviral drugs against emerging or re-emerging swine coronaviruses.

The emergence of swine acute diarrhea syndrome coronavirus (SADS-CoV), an alpha-coronavirus that causes fatal enteric disease in neonatal piglets with mortality rates up to 90%, demonstrates that bat-origin coronavirus has expanded its host range to pigs. Although SADS-CoV exhibits significant pandemic potential and capacity for cross-species transmission, the replication mechanisms of SADS-CoV remain largely unexplored. Identifying host factors responsible for SADS-CoV replication and elucidating its underlying mechanisms is essential for advancing fundamental knowledge of coronavirus biology and developing antiviral therapies. Here, we identified PABPC1 as a novel interactor of the SADS-CoV nucleocapsid (N) protein. Overexpression of PABPC1 restricted SADS-CoV infection, whereas knockdown of PABPC1 enhanced viral replication. Further study indicated PABPC1 as a host restriction factor of SADS-CoV in a manner dependent on its PABC domain. Mechanistically, PABPC1 enhances the ubiquitination of the N protein, therefore facilitating its recognition by the cargo receptor TOLLIP for selective autophagic degradation. This study systematically analyzes the interaction of host factors and the SADS-CoV N protein and identifies PABPC1 as a host restriction factor that limits viral replication via TOLLIP-mediated selective autophagy degradation of the N protein. These findings expand our knowledge of the SADS-CoV replication mechanism and provide additional antiviral strategies for controlling SADS-CoV.