Yun Lin

Background: Duck adenovirus 3 (DAdV-3) is an emerging pathogen that has caused severe economic losses to the duck industry in China. Recently, the infection of ducks with serotype 4 fowl adenovirus (FAdV-4) has also been reported in China. Therefore, an efficient bivalent vaccine to control the diseases caused by DAdV-3 and FAdV-4 is extremely urgent. In our previous study, a recombinant FAdV-4 expressing Fiber-2 of DAdV-3 was generated and designated as rFAdV-4-Fiber-2/DAdV-3. Methods: Here, the recombinant virus rFAdV-4-Fiber-2/DAdV-3 was inactivated to serve as a bivalent vaccine, and its immunogenicity and protective efficacy against DAdV-3 were evaluated in Muscovy ducks. Results: The subcutaneous injection of rFAdV-4-Fiber-2/DAdV-3 could efficiently induce antibodies against Fiber-2 of DAdV-3 and neutralize antibodies against FAdV-4. After challenges with DAdV-3, in comparison with the non-immunized ducks, the immunized ducks did not show any bodyweight loss, gross lesions, or histopathologic change. Moreover, viral loads in livers and kidneys from immunized ducks were undetectable, whereas those in non-immunized ducks with challenge were significantly high. Conclusions: All these data demonstrate that the inactivated recombinant virus rFAdV-4-Fiber-2/DAdV-3 has the potential to be an efficient vaccine candidate against both FAdV-4 and DAdV-3, although efficacy for FAdV-4 needs to be confirmed experimentally.

Duck adenovirus 3 (DAdV-3), a newly emerged duck adenovirus, has resulted in significant economic losses to the duck industry across China since 2014. However, little is known about the B cell epitopes in major antigen of DAdV-3 and the serological approach for detection of DAdV-3 is not available. In this study, four monoclonal antibodies (mAbs) specific to Fiber-2 protein of DAdV-3 were first generated and designated as 2G10, 3D9, 5E6, and 6B12. Indirect immunofluorescence assay (IFA) showed that all of the mAbs reacted with the Fiber-2. Moreover, mAbs 2G10, 5E6, and 6B12 demonstrated good activity with Fiber-2 in Western blot. Notably, the Fiber-2 could be immunoprecipitated efficiently by mAb 3D9. Epitope mapping revealed that mAbs 2G10, 3D9, 5E6, and 6B12 recognized 397-429aa, 463-481aa, 67-99aa, and 1-66aa of Fiber-2, respectively. Besides, a novel sandwich ELISA for efficient detection of DAdV-3 was developed based on mAb 3D9 and horseradish peroxidase (HRP) conjugated mAb 3D9. The sandwich ELISA only reacted with DAdV-3 but not with other duck-associated viruses. The limit of detection of the ELISA was 6.25 × 10³ TCID₅₀/mL. Overall, the mAbs generated laid the foundation for elucidating the critical role of Fiber-2 in mediating infection and pathogenesis, and the sandwich ELISA approach established here provided efficient and rapid serological diagnostic tool for DAdV-3.

Recently, the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and duck adenovirus 3 (DAdV-3) were outbroken and widespread, causing substantial economic losses to the duck industry. Therefore, there is an urgent need to generate a recombinant genetic engineering vaccine candidate against both FAdV-4 and DAdV-3. In this study, a novel recombinant FAdV-4 expressing the Fiber-2 protein of DAdV-3, designated as rFAdV-4-Fiber-2/DAdV-3, was generated based on CRISPR/Cas9 and Cre-LoxP systems. Indirect immunofluorescence assay (IFA) and western blot (WB) showed that the Fiber-2 protein of DAdV-3 in rFAdV-4-Fiber-2/DAdV-3 was expressed successfully. Moreover, the growth curve revealed that rFAdV-4-Fiber-2/DAdV-3 replicated efficiently in LMH cells and even showed a stronger replication ability compared to the wild type FAdV-4. The generation of the recombinant rFAdV-4-Fiber-2/DAdV-3 provides a potential vaccine candidate against both FAdV-4 and DAdV-3.

Duck adenovirus 3 (DAdV-3), a newly emerged duck adenovirus, has resulted in significant economic losses to the duck industry across China since 2014. However, little is known about the B cell epitopes in major antigen of DAdV-3 and the serological approach for detection of DAdV-3 is not available. In this study, four monoclonal antibodies (mAbs) specific to Fiber-2 protein of DAdV-3 were firstly generated and designated as 2G10, 3D9, 5E6, and 6B12. Indirect immunofluorescence assay (IFA) showed that all of the mAbs reacted with the Fiber-2. Moreover, mAbs 2G10, 5E6, and 6B12 demonstrated good activity with Fiber-2 in Western blot. Notably, the Fiber-2 could be immunoprecipitated efficiently by mAb 3D9. Epitope mapping revealed that mAbs 2G10, 3D9, 5E6, and 6B12 recognized 397-429aa, 463-481aa, 67-99aa, and 1-66aa of Fiber-2, respectively. Besides, a novel sandwich ELISA for efficient detection of DAdV-3 was developed based on mAb 3D9 and HRP conjugated mAb 3D9. The sandwich ELISA only reacted with DAdV-3 but not with other duck-associated viruses. The limit of detection of the ELISA was 6.25×10³ TCID₅₀/mL. Overall, the mAbs generated laid the foundation for elucidating the critical role of Fiber-2 in mediating infection and pathogenesis, and the sandwich ELISA approach established provided efficient and rapid serological diagnostic tool for DAdV-3.

Since 2015, the outbreaks of hydropericardium-hepatitis syndrome (HHS) and inclusion body hepatitis (IBH) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and serotype 8 fowl adenovirus (FAdV-8), respectively, have caused huge economic losses to the poultry industry. Although several vaccines have been developed to control HHS or IBH, a recombinant genetic engineering vaccine against both FAdV-4 and FAdV-8 has not been reported. In this study, recombinant FAdV-4 expressing the fiber of FAdV-8b, designated as FA4-F8b, expressing fiber of FAdV-8b was generated by the CRISPR-Cas9 and homologous recombinant techniques. Infection studies in vitro and in vivo revealed that the FA4-F8b replicated efficiently in LMH cells and was also highly pathogenic to 2-week-old SPF chickens. Moreover, the inoculation of inactivated the FA4-F8b in chickens could not only induce highly neutralizing antibodies, but also provide efficient protection against both FAdV-4 and FAdV-8b. All these demonstrate that the inactivated recombinant FA4-F8b generated here can act as a vaccine candidate to control HHS and IBH, and FAdV-4 can be an efficient vaccine vector to deliver foreign antigens.

Fowl adenovirus serotype 4 (FAdV-4) is the pathogenic agent of hydropericardium hepatitis syndrome (HHS) in chickens and ducks, which has caused huge economic losses for the Chinese poultry industry since 2015. In order to objectively determine the prevalence and co-infection status of the virus in Shandong province in China, we analyzed a total of 679 clinical cases of chickens and ducks from 36 farms in the province. The results showed that the FAdV-4 infection rate was 65.2% (443/679), and the rate in breeder ducks was almost two-fold higher than that in breeder chickens (68.57% vs. 34.30%). Notably, co-infection by H9N2 avian influenza virus, infectious bursal disease virus, and/or chicken infectious anemia virus was very common in the 443 FAdV-4-positive cases. Furthermore, phylogenetic analysis of the hexon genes of four Shandong FAdV-4 isolates revealed that these strains clustered into Indian reference strains, indicating that the Shandong FAdV-4 strains might have originated in India. These findings provide the first data on the prevalence and co-infection status of FAdV-4 in Shandong province, which may serve as a foundation for the prevention of FAdV-4 in the field.

Tembusu virus (TMUV) is a contagious pathogen from fowl that mainly infects ducks and geese, causing symptoms of high fever, loss of appetite, retarded growth, neurological symptoms, severe duck-drop syndrome, and even death. During an epidemiological investigation of TMUV in Northern China, we isolated 11 TMUV strains from ducks, chickens, geese, sparrows, and mosquitoes (2011–2017). Phylogenetic analysis of the open-reading frames of genes revealed that these strains clustered into Chinese strains II. The nucleotide and amino acid homologies of NS1 of the strains ranged between 85.8–99.8% and 92.5–99.68%, respectively, which were lower than those of E (86.7–99.9% and 96.5–99.9%, respectively), NS3 (87.6–99.9% and 98.2–99.8%, respectively), and NS5 (86.5–99.9% and 97.8–99.9%, respectively). Predictions of the tertiary structure of the viral proteins indicated that NS1 in 4 of 11 strains had a protein structure mutation at ¹⁸⁰TAV¹⁸² that changed a random crimp into an alpha helix. The protein of 6 of 11 strains had a glycosylation site mutation from NTTD to NITD. Furthermore, epidemiological data suggested that TMUV has been circulating in half of China’s provinces (17 of 34). Our findings, for the first time, have identified the NS1 protein as a potential hypervariable region for genetic evolution. Additionally, the territorial scope of the virus has expanded, requiring strict bio-security measures or a multivalent vaccine to control its spread.

Duck is a major waterfowl species in China, providing high-economic benefit with a population of up to 20–30 billion per year. Ducks are commonly affected by severe diseases, including egg-drop syndrome caused by duck Tembusu virus (DTMUV). The immune mechanisms against DTMUV invasion and infection remain poorly understood. In this study, duck embryo fibroblasts (DEFs) were infected with DTMUV and harvested at 12 and 24 h post-infection (hpi), and their genomes were sequenced. In total, 911 (764 upregulated and 147 downregulated genes) and 3008 (1791 upregulated and 1217 downregulated) differentially expressed genes (DEGs) were identified at 12 and 24 hpi, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that DEGs were considerably enriched in immune-relevant pathways, including Toll-like receptor signaling pathway, Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, Chemokine signaling pathway, NOD-like receptor signaling pathway, and Hematopoietic cell lineage at both time points. The key DEGs in immune system included those of the cytokines (IFN α2, IL-6, IL-8L, IL-12B, CCR7, CCL19, and CCL20), transcription factors or signaling molecules (IRF7, NF-κB, STAT1, TMEM173, and TNFAIP3), pattern recognition receptors (RIG-I and MDA5), and antigen-presenting proteins (CD44 and CD70). This suggests DTMUV infection induces strong proinflammatory/antiviral effects with enormous production of cytokines. However, these cytokines could not protect DEFs against viral attack. Our data revealed valuable transcriptional information regarding DTMUV-infected DEFs, thereby broadening our understanding of the immune response against DTMUV infection; this information might contribute in developing strategies for controlling the prevalence of DTMUV infection.