Specific pathogen-free (SFP) chickens obtained from Harbin Veterinary Institute of Chinese Academy of Agricultural Sciences (HVRI, CAAS) were housed in a negative-pressure environment at the Experimental Animal Center of HVRI. The birds had ad libitum access to water and feed. The study was approved by the Ethical and Animal Welfare Committee of HVRI (approval No. HVRI-IACUC-2021-0426-5).

The highly pathogenic FAdV-4 strain HLJFAd15 (GenBank accession number KU991797) was isolated in our laboratory (Pan et al., 2017a), and the non-pathogenic strain rHN20 was constructed as described previously (Zhang Y. et al., 2021). All FAdV-4 viruses propagated in chicken hepatocellular carcinoma (LMH) cells. The vvIBDV HLJ0504 strain was also isolated in our laboratory, and the cell-adapted IBDV rGtHLJVP2 strain, inducing cytopathic effects (CPE) in a continuous cell line of chicken embryo fibroblasts (DF-1), was constructed as described previously (Gao et al., 2011). LMH cells were cultured in DMEM/F12 (Sigma-Aldrich, St. Louis, MO, United States), whereas DF-1 in DMEM (Sigma-Aldrich). Both the mediums were supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 μ ml^–1 streptomycin, and 100 IU ml^–1 penicillin at 37°C and 5% CO₂. The anti-IBDV-VP2 monoclonal antibody was prepared in our laboratory.

The FAdV-4 rHN20 fosmid was generated from the HLJFAd15 strain. Recombinant fosmids were engineered from rHN20 in Escherichia coli DH10B cells using the Counter-Selection BAC Modification kit (Gene Bridges, Heidelberg, Germany) as described previously (Zhang Y. et al., 2021). In brief, the rpsL-neo selection antibiotic cassette was inserted into the genome-expected sites, and rpsL-neo was replaced by a PCR-amplified product or single-stranded oligonucleotide with short homology arms.

The EGFP expression cassette from the pEGFP-N1 plasmid (Clontech, Mountain View, CA, United States) was inserted into the natural 1,966-bp deletion site (Pan et al., 2018) of rHN20 to construct the rHN20-EGFP recombinant fosmid. Next, each ORF was replaced with a single-stranded oligonucleotide to construct fosmids that individually deleted the 23 ORFs in the left and right ends of the FAdV-4 genome. Finally, the adjacent ORFs in the left and right ends of the FAdV-4 genome were divided factitiously into the L1, L2, L3, R1, R2, R3, and R4 regions. Every region was first replaced with EGFP to construct fosmids, in which EGFP replaced several adjacent ORFs as described above.

VP2 from the vvIBDV strain HLJ0504 cDNA was cloned into the pEGFP-N1 plasmid, and the EGFP ORF was replaced to form the VP2-expressing cassette. The VP2 cassette was inserted into the natural1966-bp deletion site of rHN20 to obtain the rHN20-vvIBDV-VP2 fosmid.

All recombinant fosmids were transfected into LMH cells after digestion with FseI (Thermo-Fisher Scientific, Waltham, MA, United States) for 120 h post-transfection. Cells transfected with fosmids should be frozen and thawed three times, and the cell supernatant was inoculated into fresh LMH cells. The cell supernatant was collected for amplification and identification when apparent cytopathic effect (CPE) and/or green fluorescence appeared. The accuracy of all the recombinant viruses was confirmed using PCR. The VP2 expression of rHN20-vvIBDV-VP2 was identified using an indirect fluorescence antibody (IFA) assay with the monoclonal anti-IBDV-VP2.

FAdV-4 was inoculated into LMH cells at a multiplicity of infection (MOI) of 0.01. The viruses were harvested at 12, 24, 48, 72, 96, and 120 h post-infection for plaque assay to determine viral titers.

For rHN20 recombinant viruses expressing EGFP, 2-week-old SPF chickens were randomized into five groups (n = 10): a healthy control, a challenge control, and three groups, each immunized with 200 μl of rHN20, rDL3-EGFP, or r HN20-EGFP. At 14 d post-immunization, the immunized and challenge control groups were challenged with 2,000 plaque-forming units (PFU) rWT-FAdV4, whereas the healthy control group was administered DMEM/F12. Data on the morbidity and mortality of chickens were collected daily for 14 days. Viral loads were determined by collecting heart, liver, spleen, lung, kidney, thymus, and bursa from deceased or euthanized chickens at the test endpoint. The liver and thymus were used for histopathological analysis.

In addition, 50 two-week-old SPF chickens were randomized into five groups (n = 10): 2 groups were intramuscularly immunized with rHN20-vvIBDV-VP2, and the other 3 groups remained unimmunized. Neutralizing antibody titers in sera were determined at 7, 14, and 21 days post-immunization. At 21 days post-immunization, 10 immunized and 10 unimmunized chickens were challenged intramuscularly with 2,000 PFU virulent FAdV-4; the other 10 immunized and 10 unimmunized chickens were inoculated intranasally with 10⁵ EID₅₀ vvIBDV. The left 10 chickens were used as healthy control. The chickens were monitored daily, euthanized, and necropsied at 7 days post-challenge.

Total DNA from different tissues was extracted using the AxyPrep Body Fluid Viral DNA/RNA Miniprep kit (Corning, Corning, NY, United States). Virus genome copies and cellular copies were quantified using Premix Ex Taq (TaKaRa, Kusatsu, Shiga, Japan) as described previously (Baigent et al., 2005; Pan et al., 2017c). Real-time PCR was performed using QuantStudio5 (Applied Biosystems, Waltham, MA, United States). Viral loads were expressed as the number of viral genome copies per 10⁶ cells (copies 10^–6 cells) (Zhang Y. et al., 2021).

The tissues were fixed in 10% formalin and embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E) and observed under a microscope.

After inactivation at 56°C for 30 min, each serum sample was filtered through a 0.22-μm pore size filter. A twofold serial dilution of the serum was mixed with 200 median tissue culture infectious dose (TCID₅₀) of the FAdV-4 rWT-EGFP strain or IBDV rGtHLJVP2 strain in a 96-well plate, incubated at 42°C for 1 h, and then added to the LMH or DF1 cells. After being cultured at 37°C for 72 h, IBDV CPEs were observed. After being cultured at 37°C for 6 day, the GFP expressed in FAdV-4 was visualized by fluorescence microscopy.

Two-way ANOVA with Tukey’s multiple comparison test was used for identifying significant differences. Significance was set at p < 0.05. Statistical analysis was performed with GraphPad (GraphPad Software, San Diego, CA, United States).

To facilitate visualization of the virus on cells, we inserted an enhanced green fluorescent (EGFP) expression cassette on the natural 1,966 bp deletion between ORF42 and ORF43 of the FAdV-4 rHN20 strain (Figure 1A). The results showed that the rescued recombinant virus rHN20-EGFP strain could produce green fluorescence in LMH cells, whereas rHN20 could not (Figure 1B). PCR amplification of rHN20 and rHN20-EGFP genomic DNA revealed a 400-bp band for rHN20 and an approximately 2,000-bp band for rHN20-EGFP (Figure 1C). Comparing the replicative capacity of rHN20-EGFP with that of rHN20 on LMH cells, we found that rWT-EGFP replicated efficiently, despite its slightly reduced replicative capacity (Figure 1D). Thus, we successfully obtained an EGFP-expressing FAdV-4 reporter virus.

The ORFs at the left -and right-terminal regions of the FAdV genome are unique and not conserved within the Adenoviridae family (Davison et al., 2003). The FAdV-4 genome had 10 ORFs at the left end and 13 ORFs at the right end (Figures 2A, 3A). To determine whether these 23 ORFs are essential genes, they were individually deleted in the rHN20-EGFP fosmid, linearized, and transfected into LMH cells to recure ORF-knockout viruses. EGFP served as a reporter gene to monitor transfection success and viral production. At 48 h post-transfection, cells transfected with all fosmids engineered from rHN20-EGFP exhibited apparent green fluorescence, whereas mock and rWT-FAdV4 control cells showed no fluorescence (Figures 2B, 3B). Cell supernatants were harvested to infect new LMH cells, and all ORF-knockout recombinant viruses were successfully rescued (Figures 2C, 3C). Furthermore, all ORF knockout viruses were identified by PCR amplifying the knockout site fragment, resulting in a smaller band than the control (Figures 2D, 3D). Our data demonstrated that FAdV-4 could replicate after individual deletion of all 23 ORFs in both terminal regions of the viral genome.

To further confirm that the 23 ORFs in both terminal regions of the FAdV-4 genome were not essential for virus replication, the same ORFs in rHN20 were divided factitiously into L1, L2, L3, R1, R2, R3, and R4 regions that were each replaced by the EGFP cassette (Figure 4A). Seven recombinant viruses were generated, designated as rDL1-EGFP, rDL2-EGFP, rDL3-EGFP, rDR1-EGFP, rDR2-EGFP, rDR2-EGFP, and rDR3-EGFP. LMH cells infected with each of the seven recombinant viruses or rHN20-EGFP expressed green fluorescence (Figure 4B). All the eight EGFP-expressing recombinant viruses replicated efficiently in LMH cells (Figure 4C).

An immunization and challenge test was performed to evaluate whether the recombinant FAdV-4 viruses expressing EGFP could be used as live vaccines. Two-week-old SPF chickens were immunized with live rHN20, rDL3-EGFP, or rHN20-EGFP. At 14 d post-immunization, the chickens were challenged with a lethal dose of FAdV4 HLJFAd15. As described in Figure 5A, all live recombinant FAdV-4 showed 100% protection. No clinical signs or death were observed in chickens pre-inoculated with live rHN20, rDL3-EGFP, or rHN20-EGFP within 14 d post-challenge, whereas the mortality of challenge control chickens reached 80% at 4 d post-challenge (Figure 5B). Moreover, high viral loads were observed in the tissues, especially livers, of the challenge control group at 14 day post-challenge, whereas almost no viral load was detected in the tissues of the immunized groups (Figure 5C). In line with these findings, massive pathological lesions and vacuolar necrosis were observed in the liver and thymus of the challenge control group, whereas no apparent pathological changes were found in any of the rHN20-, rDL3-EGFP, or rHN20-EGFP immunized groups (Figure 5D). Therefore, the insertion of EGFP did not impair the immunogenicity of FAdV-4.

The coding region of the vvIBDV VP2 gene was inserted into the rHN20 genome to assess whether rHN20 could deliver a protective antigen after adding a CMV promoter and poly-A to rescue the recombinant virus rHN20-vvIBDV-VP2 (Figure 6A). Using PCR and IFA, we found that the vvIBDV VP2 expression cassette was precisely inserted at the natural 1,966-bp deletion, and the vvIBDV VP2 protein could be expressed (Figures 6B,C). The growth kinetics of rHN20-vvIBDV-VP2 were delayed, and the virus titer was approximately a 1-log defect compared with rHN20 (Figure 6D).

Chickens were vaccinated with rHN20-vvIBDV-VP2 to evaluate the protective efficacy of the rHN20-vvIBDV-VP2 recombinant virus, and serum neutralizing antibodies against FAdV-4 and vvIBDV were continuously detected. Next, they were challenged with FAdV-4 HLJFAd15 or vvIBDV HLJ0504 at 21 days post-vaccination. The virus neutralization assay showed that the immunized chicken serum had strong neutralizing activity against FAdV-4 (Figure 7A) and vvIBDV (Figure 7B). All immunized chickens did not show any clinical signs or mortality after the challenge with FAdV-4 (Figure 8A) or vvIBDV (Figure 8B). In contrast, challenged chickens showed high mortality (100% for FAdV-4 and 70% for vvIBDV). In line with these findings, no histopathological changes were evident in the liver or bursa of immunized chickens; however, FAdV-4 challenge controls showed hepatocyte necrosis (Figure 8C), whereas vvIBDV challenge controls showed bursa cell necrosis and follicular atrophy (Figure 8D).