Isolation, characterization, evaluation of pathogenicity, and immunomodulation through interferon production of duck adenovirus type-3 (DAdV-3)

Duck adenovirus type-3 (DAdV-3) is a poorly characterized duck virus. A comprehensive analysis of the DAdV-3 pathogenicity and host immune response could be a valuable addition. Herein, DAdV-3 was isolated from Muscovy duck and virus-specific genes were confirmed by polymerase chain reaction (PCR). The obtained gene fragments were sequenced and compared with the reference sequence. Results confirmed that the clinically isolated virus was DAdV-3, named as HF-AN-2020. To evaluate DAdV-3 host immune response, the expression levels of MDA5, STING, IRF7, MAVS, and NF-κB, and inflammatory cytokines (IFN-β, IFN-γ, and IL-1β) were determined by quantitative reverse transcriptase PCR (qRT-PCR). The expression levels of IFN-β and IFN-γ were 32.6- and 28.6-fold, respectively, higher (P < 0.01) than the control group. It was found that the upregulation of STING and NF-κB pathways was directly involved in the regulation of inflammatory cytokines (IFN-β, IFN-γ, and IL-1β). Furthermore, the gene regulation pathways consecutively upregulated the expression levels of MDA5, STING, IRF7, MAVS, and NF-κB up to 31.6, 10.5, 31.4, 2.2, and 2.6-fold, respectively, higher (P < 0.01) than the control group. The TCID50 of DAdV-3 for Muscovy duck and chicken was 10−3.24/0.1 mL with 0% mortality, indicating low pathogenicity in both Muscovy ducks and chickens, but DAdV-3 can induce higher expression of interferons. Genome analysis showed mutations in 4 amino acids located in ORF19B (Ser to Thr), ORF66 (Leu to Phe, Ile to Leu), and ORF67 (Gly to stop codon). This study provides essential and basic information for further research on the mechanism of the cellular immune responses against adenoviruses.


INTRODUCTION
According to the International Committee on Taxonomy of Viruses (ICTV), the family Adenoviridae contains 6 distinct genera (Aviadenovirus, Mastadenovirus, Atadenovirus, Siadenovirus, Ichtadenovirus, and Testadenovirus) (https://talk.ictvonline.org/)(Benk\Ho et al., 2022).The family Adenoviridae consists of several serotypes which infect many vertebrates including mammals and birds.Adenoviruses isolated from poultry are generally classified into the genus Aviadenovirus (Marek et al., 2014).Aviadenoviruses are nonenveloped, icosahedral, and double-stranded DNA, which can infect many birds such as ducks, chickens, fowl, geese, pigeons, falcons, turkeys, and psittacine species (Davison et al., 2003).Although both DAdV-2 and DAdV-3 were isolated from Muscovy ducks, they have different viral genome structures and pathogenic characteristics.The genome of DAdV-3 contains 3 main surface structural proteins including; fiber, hexon, and penton.Like the serotype 4 fowl adenovirus (FAdV-4), DAdV-3 also has 2 fiber proteins (fiber-1 and fiber-2).Previous studies have shown that the fiber proteins are related to virus neutralization, so they are ideal for the detection of viruses and vaccine development (Yin et al., 2019;Shao et al., 2022;Lin et al., 2023) The amino acid sequences of hexon proteins are different in each strain which is important for virus classification (Marek et al., 2010).Penton, as a bond between hexon and fiber, plays an important role in the stability of viral capsid and the attachment of invading virion (Gao et al., 2019).
Several molecular methods such as polymerase chain reaction (PCR), real-time PCR, high resolution melting (HRM) curve analysis, and loop-mediated isothermal amplification (LAMP) have been used for the detection of aviadenoviruses based on the major capsid protein (hexon) or the DNA-dependent DNA polymerase (Kaj an et al., 2011;Xie et al., 2011;G€ unes et al., 2012).DAdV-3 (GD-CH-2014) was first discovered in China in 2014.The ducks infected with DAdV-3 were characterized by swelling, bleeding, and hemorrhages of the liver and kidneys, with a morbidity rate of 40 to 55% and a mortality rate of 35 to 43%, which caused huge economic losses to the duck industry (Zhang et al., 2016;Yin et al., 2022).However low virulence strains of DAdV-3 have not been reported so far.
In this study, DAdV-3 was isolated and identified from the Muscovy duck farm, and its pathogenicity to Muscovy ducks and chickens was analyzed.The possible immune mechanism was explored to pave for further research on its prevention and control.

Animals and Ethics Statement
Day-old specific-pathogen-free (SPF) chick and chick embryos were purchased from Zhejiang Lihua Agricultural Co., Ltd.(Anhui, China).Muscovy ducks were procured from Yongqiang Agricultural Co., Ltd.(Anhui, China).These birds were kept in a healthy and controlled environment for 30 d.The temperature was maintained according to the age and behavior of the birds (28°C−33°C).All birds were kept and handled according to the Institutional Animal Care and Use Committee (IACUS) guidelines by the School of Animal Science and Technology, Anhui Agricultural University, Hefei, China (AHAU 2020-010).

DAdV-3 (HF-AH-2020) Isolation
The liver tissues of a 30-day-old DAdV-3 infected Muscovy duck were collected from the Avian Disease Diagnostic Center of Anhui Agricultural University, Hefei, China, and used as a DAdV-3 source for this study.The liver was cut into pieces and grounded into a homogenate mixture under sterile conditions.Subsequently, Hank's solution (Beyotime, Shanghai, China) was added to this homogenate mixture to make a 10% suspension.After this suspension was frozen and thawed 3 times, then centrifuged at 12,000 rpm for 30 min at 4°C . The supernatant was filtered through a 0.22 mm filter and stored at À80°C for further use.
The supernatant was inoculated with the dose rate of 0.15 mL/embryo into the allantoic cavity of 8-day-old SPF chicken embryos.The inoculated embryos were incubated at 37°C with a relative humidity of 55%.The embryos that died within 2 d were discarded, whereas those that died between 9 and 10 d were stored for the collection of DAdV-3.

DAdV-3 (HF-AH-2020) Nucleic Acid Extraction and Phylogenetic Analysis
The total RNA and DNA were extracted by viral RNA/DNA purification kit (Tiangen, Beijing, China) according to the manufacturer's instructions.RNA was reverse-transcribed into cDNA according to manufacturer instruction (Tiangen, Beijing, China), and used as a PCR template.The hexon gene of adenovirus was amplified by a self-designed primer: Hexon F: ATGGCCGCTCTGACCCCTGA and Hexon R: ATT-CAGCCTTAGCTACTTTC.In addition to DAdV-3, the same samples were tested for other viruses such as; duck stellate virus (DAstV), duck Tembusu virus (DTMUV), avian pulmonary virus (AMPV), avian influenza viruses H5 and H7 (AIV-H5 and AIV-H7), and duck circovirus (DuCV).Optimized primers from other studies were also used in our experiments (Liu et al., 2016;Weiguo et al., 2016;Yu et al., 2019;Huang et al., 2020).The viral nucleic acid amplification and identification was done by PCR (G€ unes et al., 2012;Pan et al., 2017), and amplified DNA fragments were cloned into a pMD18-T vector (Dongsheng, Guangzhou, China) for sequencing (Huada, Shanghai, China).The obtained hexon gene sequences were aligned for gene analysis.Furthermore, 26 avian adenovirus strains; FAdV-A, B, C, D, E, and DAdV were selected for the phylogenetic analysis of hexon protein.The amino acid sequences were aligned by using BioEdit v7.2.5.The best amino acid substitution models were MEGA v6.0.and DNA star, used for evolutionary genetic analysis of the target gene using the maximum likelihood (ML) method, to obtain the phylogenetic tree of the isolated strain (DAdV-3).

Cytopathic Effect (CPE) of DAdV-3 (HF-AH-2020)
Chicken embryonic kidney (CEK) cells were cultured according to previously reported methods (Li et al., 2018(Li et al., , 2021)).CEK cells were inoculated in 6 well plates with a ratio of 1 £ 10 7 /well overnight.After overnight incubation, the supernatant of these cultured cells was discarded, 500 mL serum-free RPMI medium (Hyclone, South Logan, Utah) modified with 10 mL allantoic fluid of Muscovy duck, and 30 mL allantoic fluid of chicken embryo was added to each well, then incubated for 1 to 2 h, and last 2 wells were used as negative control (only PBS).Subsequently, the supernatant was discarded, RPMI Medium Modified with 1% FBS was added, and changes in the cells were observed daily.
RT-PCR-Based Organ-Specific Detection of DAdV-3 (HF-AH-2020) To analyze the distribution of the virus in different organs (heart, liver, spleen, kidney, brain, forestomach, glandular stomach, and intestine) of chicken embryos, 3 embryos within an hour of death were used to collect the 50 mg tissue sample taken from each above-mentioned organs for DNA extraction.The primers for DAdV-3 were designed for the highly conserved region of the DAdV-3 52K gene.The primers sequences were followed as 52k-F: 5 0 -TGTGGAAATCGGGGTGTATC-3 0 , 52k-R: 5 0 -GTCAAACCGAACATGTAGTCTG-3 0 .The plasmid pMD18-T-52K was also designed.The quantification of DAdV-3 through real-time PCR (RT-PCR) was done by using AceQ qPCR SYBR Green Master Mix (Vazyme, China).The standard curves were obtained from 3 independent experiments each performed in duplicates by using 10-fold serial dilutions of plasmid (pMD18-T-52 K) and its identification was done by RT-PCR.The number of viral copies per gram was calculated by comparing the values of the threshold cycle (CT) with standard curve values.Each reaction was performed in triplicate, to get mean § standard deviation (SD).The calculated values of viral load were expressed as the number of viral copies per gram of tissues (No.VC/g tissue).

Amplification and Structural Analysis of DAdV-3 (HF-AH-2020) Whole Genome
The viral DNA was extracted from the liver of DAdV-3 infected Muscovy duck by using a DNA extraction kit (Anheal, Beijing, China).Genomic DNA was quantified by using a TBS-380 fluorometer (Turner Bio-Systems Inc., Sunnyvale, CA).The high-quality DNA samples (OD260/280 = 1.8−2.0,>6 ug) were used to construct a library of DNA fragments.The complete viral genome was sequenced by Illumina Hiseq combined with third-generation sequencing technology.Sequencing and genome assembly was performed by Shanghai Biozeron Biotechnology Co, Ltd. (Shanghai, China).The genome sequence was submitted to NCBI, and the nucleotide sequence was compared to the NCBI GenBank database, using the BLAST Search Tool (BLASTn).The amino acid sequences of the putative proteins were also compared to proteins, present in the NCBI GenBank database by using the protein (BLASTp).Moreover, Snapgene 4.2.4 software was also used to compare and analyze the sequences of amino acids isolated with a virulent strain of DAdV-3 (GD-CH-2014).

DAdV-3 (HF-AN-2020) Virus Titration
Chicken hepatocellular carcinoma (LMH) cells were cultured in a culture medium containing 10% FBS (GE, Utah) and DMEM/F12 (Biosharp, Beijing, China).This media was spread in 96-well culture plates at the ratio of 1 £ 10 4 cells/100 mL.These plates were incubated overnight with 5% CO 2 at 37°C.The virus was diluted 10fold (1/10) with PBS, followed by 8 gradients of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , and 10 8 in the total volume of 100 mL/well, and the last 2 wells were kept as negative control (only PBS).After incubation at 37℃ for 2 h, the culture media of all wells were replaced with a 0.1 mL culture medium containing 1% FBS for incubation for up to 9 d.During culture, the characteristics of infected cells were; round, aggregation, enhanced refractive index, and typical beads on a string.The period of infection and death of the cells was recorded as 50% Tissue Culture Infective Dose (TCID 50 ) according to the Reed-Muench formula (Matumoto, 1949).

Isolation of Bacteria and Identification of Virulence Island
In order to verify whether the isolated HF-AN-2020 strain was coinfected with bacteria or not, bacteria were isolated and identified from the liver of a Muscovy duck at the same time.The liver of the Muscovy duck was dissected under aseptic conditions and cultured in a TSB medium (BD, Maryland) at 37°C with 5% CO 2 for 48 h.After this, selecting 1 colony for Gram staining and PCR identification of bacterial 16sRNA was done.The preliminarily identified E. coli was amplified and recognized for the virulence island gene.The identification methods of bacteria and their virulence was performed according to the previously used method (Wang et al., 2015).

Animal Trials
Fourteen-day-old SPF chickens and Muscovy ducks were randomly divided into 8 groups, 15 in each group.The 0.25 mL virus (HF-AN-2020) was introduced by intramuscular (IM) injection in the thigh muscles of chickens and Muscovy ducks with a dose rate of 10 À3.24 TCID 50 /0.1 mL for the positive control groups whereas normal saline was injected in the negative control group with the same dose rate (0.25 mL of PBS).All birds were kept according to the standard protocols of IACUS and monitored for 12 d of postinfection.After fourth day of infection, cloacal swabs were collected from 3 chickens and 3 ducks in each group, and livers were extracted for virus detection after the euthanization of the birds.Meanwhile, the liver and kidney samples were collected from each group and sent to Servicebio Biotechnology Co, Ltd. (Wuhan, China) for histopathological analysis.On the 12th day of postinfection, 0.5 mL blood was drawn from the wing veins of both birds (chickens and ducks) via a syringe and serum was separated for the detection of antibody titers.

Enzyme-Linked Immunosorbent Assay
The indirect enzyme-linked immunosorbent assay (ELISA) method was used to determine the DAdV-3-specific antibody titers in the serum of all experimental birds.Twenty mL of allantoic fluid was collected from infected (DAdV-3) chicken embryos.For the isolation of DAdV-3, the infected allantoic fluid was centrifuged at 28,000 rpm for 4.5 h to obtain 1 mL of concentrated allantoic fluid.The protein concentration was detected with the BCA protein concentration assay kit (Beyotime, Shanghai, China).Hundred mL of concentrated DAdV-3 (HF-AH-2020) (1 mg/mL) was placed into each well of the ELISA plates and incubated overnight at 4 ℃.After incubation, the plates were washed 3 times with PBS containing 0.05% Tween-20 (PBST) and then incubated with 5% FBS (Beyotime, Shanghai, China) for 1 h at 37°C.After 3 washes, chicken or duck serum samples were diluted with a ratio of 1:20,000 and again incubated at 37°C for 1 h.The following incubated samples were again washed 3 times with PBST and incubated for 1 h at 37°C with HRP-conjugated goat Anti-Bird IgY (Abcam) via dilution rate of 1:5,000.After washing, 100 mL tetramethylbenzidine substrate (Beyotime, Shanghai, China) was added to each well, and the plates were incubated in the dark for 10 min.The enzymatic reaction was quenched by hydrofluoric acid, and the optical density (OD) was determined at 450 nm.Meanwhile, The serum of negative chickens or ducks was negative control.Sample value was calculated as a ratio using the formula: value = ODsample À ODneg.Each sample was analyzed in triplicate.

Detection of Interferon b and g Genes Expression by qRT-PCR
To investigate the reason for the low pathogenicity of DAdV-3 (HF-AH-2020), we detected the expression of interferons and related genes after 72 h of infection with CEK cells.The experiment of CEK cells infected with DAdV-3 (HF-AH-2020) was repeated 3 times.The reference sequence of cytokines (IFN-b, IFN-g, and IL-1b), signaling pathways regulating molecules NF-kB and stimulator of interferon genes (STING), membranerelated receptor (MDA5), interferon regulatory factor-7 (IRF7) and signal protein (MAVS) genes were used for primer designing via GenBank.The names of the genes, primer sequences, amplified fragment size, and login ID numbers have been taken from previous studies (Li et al., 2021).
Cytokines (IFN-b, IFN-g, and IL-1b), signaling pathways regulating molecules (NF-kB, STING), membrane-related receptor (MDA5), signal protein IRF7 and MAVS were detected by using cDNA as a template through real-time reverse transcription-PCR (qRT-PCR).At the same time, FAdV-4 infected cells were used as a control group, and relative expression was calculated by the 2 ÀDDCT method.

Statistical Analysis
The statistical analysis was performed by SPSS 16.0 (SPSS Inc., Chicago, IL).The results of all experiments were analyzed by t test, followed by Tukey's honestly significant differences (HSD) for posthoc testing to compare the significance (P) between the means of different groups.The differences were considered statistically significant at P < 0.01.

Whole Genome Sequence Analysis of DAdV-3 (HF-AH-2020)
The complete genome sequence of a DAdV-3 was obtained by sequencing the whole genome, and complete amplified genome DAdV-3 (HF-AH-2020) sequencing data were submitted to the GenBank, under the accession number MT792736.The whole genome sequencing analysis showed that DAdV-3 nucleotides were homologous to DAdV-2 and DAdV-4 with 92.3 and 57.2%, respectively.As shown in Figure 3, the main structural proteins of DAdVs were include 52K, pII (hexon), pIII (penton), DBP (DNA binding protein), 100K, and fiber.DAdV-3 (MT792736) and DAdV-4 (MN733730) had 2 fibers (fiber-1 and fiber-2), but the locations of these both fibers (proteins) were different in different viral genomes, while DAdV-2 (KJ469653) has only 1 fiber (Figure 3).Snapegne analysis shows that the main amino acid changes in HF-AH-2020 and GD-CH-2014 were ORF19B(Ser to Thr), ORF66(Leu to Phe, Ile to Leu), and ORF67(Gly to stop codon), and there were 1 to 2 amino acid mutations in each of these ORFs, among which the mutation of ORF67 lead to early termination of ORF67 protein translation.

Distribution of DAdV-3 (HF-AN-2020) in Embryonic Stage
SPF chicken embryos were died within 9 to 10 d after DAdV-3 inoculation.The DAdV-3 infected chicken embryos were short and stunted, and the whole-body surface was covered with a large number of petechial hemorrhages.Further, punctate necrosis was observed on the surface of the liver, while it was a normal texture in the control group (Figure 4).Three embryos within an hour of death were used to collect the tissues (50 mg) from each organ such as; the heart, liver, spleen, kidney, brain, forestomach, glandular stomach, and intestine.The tissues of these organs were used to extract the viral (DAdV-3) DNA and the viral load in each organ was determined by RT-PCR.The recombinant plasmid (pMD18-T-52k) was constructed with the target gene Table 1.Homology comparison of DAdV-3 genome with different DAdVs domains.

Cellular Changes Caused by DAdV-3 (HF-AH-2020) Infection
CEK cells were used to identify changes in DAdV-3 infection.CEK cells were infected with the supernatant with 10 mL grinded liver tissues and 30 mL of allantoic fluid to observe the pathological changes (shrinking, clustering, and death) in CEK cells which were started from d 4 and gradually increased up to d 7. The infected cells showed poor refractive index, turned round, and became clustered like grapes, while on the other hand, the control negative cells remained normal (Figure 6).
The TCID 50 of the DAdV-3 (HF-AH-2020) DAdV-3 caused cytopathic effect (CPE) in LMH cells during in vitro culture in 96-well plates.The cultured DAdV-3 was diluted 10 times and then used to infect the LMH cells in a 96-well cell culture plate to observe the cell changes.After 9 d of culture, the TCID 50 of the virus calculated according to the Reed-Muench method was found to be 10 À3.24 /0.1 mL.

Isolation of Bacteria and Identification of Virulence Island
Isolation and identification of bacteria from Muscovy duck liver were done by Gram staining and bacterial 16sRNA.The results of Gram staining and bacterial 16sRNA showed that the isolated bacteria was Escherichia coli with 5 virulence island genes including Tsh (temperature-sensitive hemagglutinin), Cav (structural genes of colicin V operon), lss (increased serum survival), Irp2 (iron-repressible protein), and lucd (aerobactin synthesis).

Necropsy Findings and Level of Antibodies
On the 7th and 14th day of age, all birds (chickens and ducks) were infected with DAdV-3 (HF-AH-2020) for 3 consecutive days (0.25 mL/d).No mortality was observed within 12 d of postinfection (Table 2).On fourth day after the infection, there were no obvious gross pathological and histopathological changes in livers and kidneys.The cloacal swab and liver samples were collected to detect the DNA of HF-AH-2020.On the 12th day of postinfection, 0.5 mL blood was drawn from the wing veins of both birds (chickens and ducks) via a syringe and serum was separated for the detection of antibody titers.When the experimental chicken and duck serum dilution reached 1:20,000, the OD450 nm value ranged from 2.32 to 2.93, while the control group ranged from 0.13 to 0.2, and there was no significant difference between chickens and duck antibody titers.Notably, the serum DAdV-3 antibody levels were higher in the infected birds than in the control group, with a positive rate of 100%.
DAdV-3 (HF-AH-2020) Induced IFN-b and IFNg Production in Chicken Embryonic Kidney Cells IFN-b and IFN-g are immunomodulatory and antiviral cytokines that play an important role in host antiviral response.To understand the transcription levels of interferon in infected cells, IFN-b and IFN-g expression levels were determined by qRT-PCR.The expression level of the IFN-b and IFN-g genes in DAdV-3infected cells was significantly higher than the FAdV-4 infected group 3 days postinfection (Figure 7).The gene expression of IFN-b in FAdV-4 and DAdV-3 infection was increased by 7.9 and 32.6 times respectively while the expression level of IFN-g in FAdV-4 and DAdV-3 was increased by 22.8 and 28.6 times, respectively.The statistical analysis showed that the gene expression of IFN-b and IFN-g was significantly different from the control group (P < 0.01), indicating that the ability of DAdV-3 to induce interferon was stronger than FAdV-4.

Cellular Pathway of IFN-b and IFN-g Production
To explore the signaling pathway, the DAdV-3 stimulates the cells to produce interferons identified in this study.We verified that the expression level of MDA5, STING, IRF7, and MAVS rises to 31.6, 10.5, 31.4,and 2.2 times, respectively, compared to the control group (P < 0.01) (Figure 8A).The virus-stimulated CEK cells significantly increased the expression level of NF-kB and IL-1b up to 2.6 and 16.3 times respectively than the control group (P < 0.01) (Figure 8B).The result suggested that DAdV-3 could induce IFN-b and IFN-g gene expression in the cells to enhance the immune response by STING and NF-kB pathways.

Figure 2 .
Figure 2. Phylogenetic analysis of DAdV-3 (HF-AH-2020) hexon gene with different serotypes of adenoviruses.The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model.The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown below the branches.Evolutionary analyses were conducted in MEGA6.0 software.

Figure 4 .
Figure4.DAdV-3 infected chicken embryos were showing short and stunted growth, the whole-body surface was covered with a large number of petechial hemorrhages, and the liver showed punctate necrosis, while the control negative group (PBS) was normal.

Figure 5 .
Figure5.The DAdV-3 (HF-AN-2020) viral load was higher in the liver, whereas the other organs' viral load sequence was the spleen, duodenum, kidney, glandular stomach, heart, muscle, muscular stomach, and brain.

Figure 6 .
Figure 6.Cytopathic effects of DAdV-3 (HF-AH-2020) on CEK cells can see clearly; the morphology of infected CEK cells was changed from normal to round and clusters like grapes.(A) CEK cells Infected by 30 mL grinded liver solution of infected Muscovy duck.(B) CEK cells infected via allantoic fluid of infected chicken embryo.(C) and (D) CEK cells infected with PBS (negative control).The pictures were taken at 72 h after inoculation by using the scale bar of 100 mm.

Figure 7 .
Figure7.The expression levels of IFN-b and IFN-g genes in CEK cells infected with DAdV-3 (HF-AN-2020) were significantly higher than the FAdV-4.Seventy-two hours of postinfected CEK cells were collected for detecting the expression of IFN-b and IFN-g genes using qRT-PCR analysis.ÃÃ Indicate the highly significant difference (P < 0.01).

Table 2 .
Test results of infected SPF chicken and Muscovy duck.