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Abstract Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N69, N78, and N265) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease.

Abstract During the replication process, the herpesvirus genome forms the head-to-tail linked concatemeric genome, which is then cleaved and packaged into the capsid. The cleavage and packing process is carried out by the terminase complex, which specifically recognizes and cleaves the concatemeric genome. This process is governed by a cis-acting sequence in the genome, named the a sequence. The a sequence and genome cleavage have been described in some herpesviruses, but it remains unclear in duck plague virus. In this study, we analysed the location, composition, and conservation of a sequence in the duck plague virus genome. The structure of the DPV genome has an a sequence of (DR4)m-(DR2)n-pac1-S termini (32 bp)-L termini (32 bp)-pac2, and the length is 841 bp. Direct repeat (DR) sequences are conserved in different DPV strains, but the number of DR copies is inconsistent. Additionally, the typical DR1 sequence was not found in the DPV a sequence. The Pac1 and pac2 motifs are relatively conserved between DPV and other herpesviruses. Cleavage of the DPV concatemeric genome was detected, and the results showed that the DPV genome can form a concatemer and is cleaved into a monomer at a specific site. We also established a sensitive method, TaqMan dual qRT‒PCR, to analyse genome cleavage. The ratio of concatemer to total viral genome was decreased during the replication process. These results will be critical for understanding the process of DPV genome cleavage, and the application of TaqMan dual qRT‒PCR will greatly facilitate more in-depth research.

Abstract Duck Tembusu virus (DTMUV), an emerging pathogenic flavivirus, causes markedly decreased egg production in laying duck and neurological dysfunction and death in ducklings. Vaccination is currently the most effective means for prevention and control of DTMUV. In previous study, we have found that methyltransferase (MTase) defective DTMUV is attenuated and induces a higher innate immunity. However, it is not clear whether MTase-deficient DTMUV can be used as a live attenuated vaccine (LAV). In this study, we investigated the immunogenicity and immunoprotection of N7-MTase defective recombinant DTMUV K61A, K182A and E218A in ducklings. These three mutants were highly attenuated in both virulence and proliferation in ducklings but still immunogenic. Furthermore, a single-dose immunization with K61A, K182A or E218A could induce robust T cell responses and humoral immune responses, which could protect ducks from the challenge of a lethal-dose of DTMUV-CQW1. Together, this study provides an ideal strategy to design LAVs for DTMUV by targeting N7-MTase without changing the antigen composition. This attenuated strategy targeting N7-MTase may apply to other flaviviruses.

Abstract Duck hepatitis A virus type 1 (DHAV-1) is an acute, highly lethal infectious agent that infects ducklings and causes up to 95% mortality in ducklings up to 1 week of age, posing a significant economic threat to the duck farming industry. Previous studies have found that the proteolytic enzyme 3 C encoded by DHAV-1 can inhibit the IRF7 protein from blocking the upstream signaling pathway of the type I interferon to promote viral replication. However, there are still few studies on the mechanism of DHAV-1 in immune evasion. Here, we demonstrate that the DHAV-1 3CD protein can interact with IRF7 protein and reduce IRF7 protein expression without directly affecting IRF7 protein nuclear translocation. Further studies showed that the 3CD protein could reduce the expression of RIG-I protein without affecting its transcription level. Furthermore, we found that the 3CD protein interacted with the N-terminal structural domain of RIG-I protein, interfered with the interaction between RIG-I and MAVS, and degraded RIG-I protein through the proteasomal degradation pathway, thereby inhibiting its mediated antiviral innate immunity to promote DHAV-1 replication. These data suggest a novel immune evasion mechanism of DHAV-1 mediated by the 3CD protein, and the results of this experiment are expected to improve the understanding of the biological functions of the viral precursor protein and provide scientific data to elucidate the mechanism of DHAV-1 infection and pathogenesis.

Abstract An alphaherpesvirus carries dozens of viral proteins in the envelope, tegument and capsid structure, and each protein plays an indispensable role in virus adsorption, invasion, uncoating and release. After infecting the host, a virus eliminates unfavourable factors via multiple mechanisms to escape or suppress the attack of the host immune system. Post-translational modification of proteins, especially phosphorylation, regulates changes in protein conformation and biological activity through a series of complex mechanisms. Many viruses have evolved mechanisms to leverage host phosphorylation systems to regulate viral protein activity and establish a suitable cellular environment for efficient viral replication and virulence. In this paper, viral protein kinases and the regulation of viral protein function mediated via the phosphorylation of alphaherpesvirus proteins are described. In addition, this paper provides new ideas for further research into the role played by the post-translational modification of viral proteins in the virus life cycle, which will be helpful for understanding the mechanisms of viral infection of a host and may lead to new directions of antiviral treatment.

Abstract Duck hepatitis A virus type 1 (DHAV-1) is one of the main pathogens responsible for death in ducklings. Autophagy is a catabolic process that maintains cellular homeostasis, and the PI3KC3 protein plays an important role in the initiation of autophagy. DHAV-1 infection induces autophagy in duck embryo fibroblasts (DEFs) but the molecular mechanism between it and autophagy has not been reported. First, we determined that DHAV-1 infection induces autophagy in DEFs and that autophagy induction is dependent on the integrity of viral proteins by infecting DEFs with UV-inactivated or heat-inactivated DHAV-1. Then, in experiments using the pharmacological autophagy inducer rapamycin and the autophagy inhibitor chloroquine, autophagy inhibition was shown to reduce intracellular and extracellular DHAV-1 genome copies and viral titres. These results suggest that autophagy activated by DHAV-1 infection in DEFs affects DHAV-1 proliferation and extracellular release. Next, we screened the autophagy-inducing effects of the DHAV-1 structural proteins VP0, VP3, and VP1 and found that all DHAV-1 structural proteins could induce autophagy in DEFs but not the full autophagic flux. Finally, we found that VP1 promotes protein expression of PI3KC3 and Beclin1 by western blot experiments and that VP1 interacts with PI3KC3 by co-immunoprecipitation experiments; moreover, 3-MA-induced knockdown of PI3KC3 inhibited VP1 protein-induced autophagy in DEFs. In conclusion, the DHAV-1 structural protein VP1 regulates the PI3KC3 complex by interacting with PI3KC3 to induce autophagy in DEFs.

Abstract Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are cytosolic pattern recognition receptors that initiate innate antiviral immunity. Recent reports found that duck RLRs significantly restrict duck plague virus (DPV) infection. However, the molecular mechanism by which DPV evades immune responses is unknown. In this study, we first found that the DPV UL41 protein inhibited duck interferon-β (IFN-β) production mediated by RIG-I and melanoma differentiation-associated gene 5 (MDA5) by broadly downregulating the mRNA levels of important adaptor molecules, such as RIG-I, MDA5, mitochondrial antiviral signalling protein (MAVS), stimulator of interferon gene (STING), TANK-binding kinase 1 (TBK1), and interferon regulatory factor (IRF) 7. The conserved sites of the UL41 protein, E229, D231, and D232, were responsible for this activity. Furthermore, the DPV CHv-BAC-ΔUL41 mutant virus induced more duck IFN-β and IFN-stimulated genes (Mx, OASL) production in duck embryo fibroblasts (DEFs) than DPV CHv-BAC parent virus. Our findings provide insights into the molecular mechanism underlying DPV immune evasion.

Abstract Fowl cholera caused by Pasteurella multocida exerts a massive economic burden on the poultry industry. Lipopolysaccharide (LPS) is essential for the growth of P. multocida genotype L1 strains in chickens and specific truncations to the full length LPS structure can attenuate bacterial virulence. Here we further dissected the roles of the outer core transferase genes pcgD and hptE in bacterial resistance to duck serum, outer membrane permeability and virulence in ducks. Two P. multocida mutants, ΔpcgD and ΔhptE, were constructed, and silver staining confirmed that they all produced truncated LPS profiles. Inactivation of pcgD or hptE did not affect bacterial susceptibility to duck serum and outer membrane permeability but resulted in attenuated virulence in ducks to some extent. After high-dose inoculation, ΔpcgD showed remarkably reduced colonization levels in the blood and spleen but not in the lung and liver and caused decreased injuries in the spleen and liver compared with the wild-type strain. In contrast, the ΔhptE loads declined only in the blood, and ΔhptE infection caused decreased splenic lesions but also induced severe hepatic lesions. Furthermore, compared with the wild-type strain, ΔpcgD was significantly attenuated upon oral or intramuscular challenge, whereas ΔhptE exhibited reduced virulence only upon oral infection. Therefore, the pcgD deletion caused greater virulence attenuation in ducks, indicating the critical role of pcgD in P. multocida infection establishment and survival.

Abstract Duck enteritis virus (DEV) is a member of the Alphaherpesvirinae subfamily. The characteristics of some DEV genes have been reported. However, information regarding the DEV UL47 gene is limited. In this study, we identified the DEV UL47 gene encoding a late structural protein located in the nucleus of infected cells. We further found that two domains of DEV pUL47, amino acids (aa) 40 to 50 and 768 to 777, could function as nuclear localization sequence (NLS) to guide the nuclear localization of pUL47 and nuclear translocation of heterologous proteins, including enhanced green fluorescent protein (EGFP) and beta-galactosidase (β-Gal). Moreover, pUL47 significantly inhibited polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced interferon beta (IFN-β) production and downregulated interferon-stimulated gene (ISG) expression, such as Mx and oligoadenylate synthetase-like (OASL), by interacting with signal transducer and activator of transcription-1 (STAT1).