Rapid Construction of an Infectious Clone of Fowl Adenovirus Serotype 4 Isolate

Adenovirus vectors possess a good safety profile, an extensive genome, a range of host cells, high viral yield, and the ability to elicit broad humoral and cellular immune responses. Adenovirus vectors are widely used in infectious disease research for future vaccine development and gene therapy. In this study, we obtained a fowl adenovirus serotype 4 (FAdV-4) isolate from sick chickens with hepatitis–hydropericardium syndrome (HHS) and conducted animal regression text to clarify biological pathology. We amplified the transfer vector and extracted viral genomic DNA from infected LMH cells, then recombined the mixtures via the Gibson assembly method in vitro and electroporated them into EZ10 competent cells to construct the FAdV-4 infectious clone. The infectious clones were successfully rescued in LMH cells within 15 days of transfection. The typical cytopathic effect (CPE) and propagation titer of FAdV-4 infectious clones were also similar to those for wild-type FAdV-4. To further construct the single-cycle adenovirus (SC-Ad) vector, we constructed SC-Ad vectors by deleting the gene for IIIa capsid cement protein. The FAdV4 infectious clone vector was introduced into the ccdB cm expression cassette to replace the IIIa gene using a λ-red homologous recombination technique, and then the ccdB cm expression cassette was excised by PmeI digestion and self-ligation to obtain the resulting plasmids as SC-Ad vectors.


Introduction
Fowl adenovirus (FAdV) belongs to the Aviadenovirus genus in the family Adenoviridae. FAdV can be grouped into five species (FAdV-A to FAdV-E) based on their molecular structure, and 12 serotypes (FAdV-1 to 8a and 8b to 11) have been identified based on their serological relationships [1,2]. Fowl adenovirus serotype 4 (FAdV-4) has been grouped with the FAdV-C species; it is highly pathogenic to chickens, particularly 3-5-week-old broilers. It is a double-stranded DNA virus with a genome of approximately 45 kb and encodes 10 major structural proteins and 11 nonstructural proteins [3][4][5][6]. FAdV-4 causes hydropericardium syndrome (HPS), inclusion body hepatitis (IBH), respiratory tract disease, and gizzard erosion in chickens [7][8][9][10][11]. Severe disease in broiler chickens is characterized by the accumulation of clear, straw-colored fluid in the pericardial sac, as well as nephritis and hepatitis [12,13]. Since 2015, outbreaks of novel FAdV-4 genotype infections have led to considerable financial losses in the poultry sector in China.
Given their strong gene expression, high titers, and ability to induce an immunological response, adenoviral (Ad) vectors have demonstrated potential as vaccine platforms. [14,15]. With the advancements of reverse genetic technology and gene editing, several viruses have been successfully modified into viral vectors, including adenovirus [16], pox-virus [17], etc. Adenovirus vectors are widely applied in gene therapy [18], as well as in vaccine development against emergent viruses such as SARS-CoV-2 [19], MERS-CoV [20], and

Plasmid and Cell
LMH cells (ATCC CRL-2117) were cultured with DMEM medium containing 10% fetal bovine serum (FBS) at 37 • C in a 5% CO 2 incubator. pFPAV3 and pKD46 were preserved in our laboratory.

Virus Isolation
During an HHS epidemic at a poultry farm in Shaanxi Province, China, liver samples were collected from a deceased chicken. Samples were mixed with phosphate-buffered saline (PBS), sheared, and ground into a liquid form. The viral solution was subjected to three rounds of the freeze-thaw procedure and was centrifuged at 4 • C, 15,292× g for 10 min. The viral solution was filtered with a 0.22 µm microporous filter and inoculated into 6-8-day-old SPF chicken embryos incubated in a 37 • C incubator. The embryos were candled every 12 h and dead embryos discarded within 24 h. After 9 days, allantoic fluid was extracted. The freeze-thawing of allantoic fluid was repeated 3 times, followed by centrifugation to remove cell debris: 15,292× g for 10 min. The above allantoic fluid was blindly passed on embryonated chicken eggs three times. The allantoic fluid was used as the seed virus to infect LMH cells incubated at 37 • C in 5% CO 2

. The cytopathic effect (CPE)
Viruses 2023, 15, 1657 3 of 12 of LMH cells was observed at 36, 72, and 96 h after infection. The culture was freeze-thawed in three rounds, cycling from 37 • C to −80 • C. To remove cell debris, centrifugation was performed at 15,292× g for 10 min, and the supernatant was cryopreserved at −80 • C.

PCR Amplification and Sequencing of the Hexon Gene
Utilizing a modified version of the traditional genome extraction approach, adenoviral genomic DNA was recovered from viral culture [36] and used as a template to amplify the partial hexon gene sequence of FAdV-4 via PCR using specific hexon-F and hexon-R primers [37] (Table A1). The amplified product was visualized using 1% agarose gel electrophoresis and purified using the Gel Extraction Kit (TIANquick Midi Purification Kit, Beijing, China). The purified PCR products were ligated into a pMD19-T plasmid, which was transformed into Top10 competent cells. The cells were spread on ampicillin-containing LB agar plates and incubated at 37 • C for 12 h. The positive colonies were picked and grown in ampicillin-containing liquid LB medium at 37 • C with shaking for 6 h, validated by colony PCR, and subjected to direct Sanger sequencing by Beijing Tsingke Biotech Co., Ltd. (Beijing, China).

Hexon Gene Sequence Analysis and Phylogenetic
The nucleotide sequence of the hexon gene of the FAdV isolate was compiled using SnapGene. The 18 reference strains of FAdV were retrieved from the NCBI GenBank database. The phylogenetic tree was built using the neighbor-joining method by MEGA 7.0.

Animal Regression Test
Twenty chicks hatched from the eggs of white specific pathogen-free (SPF) broilers acquired from the Animal Experimental Centre of Yangling Lvfang Biological Engineering Co., Ltd., Yangling, China, were raised in an isolator. At twenty days of age, ten of these chicks were intramuscularly injected with third-passage viruses from LMH cells (0.2 mL per chicken, virus titer 1 × 10 7 TCID 50 /mL) maintained in LMH cells, and the other chicks were intramuscularly injected with the same dose of physiological saline for use as the negative control. Clinical symptoms and pathological changes were observed and recorded 3 times per day. The dead chickens were quickly dissected, and pathological changes in heart and liver tissue were observed. After 14 days of continuous observation, the surviving chickens were dissected, and the results were recorded.

Construction of the Recombinant pShuttle-FAdV-4 Plasmid with the DNA Assembly Technique
First, using the swine adenovirus type 3 infectious clone(pFPAV3) as a template, the primers (pShuttle-PacI-ITR-F/pShuttle-PacI-ITR-R) (Table A1) flanked by the partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing pShuttle-FAdVITR-FR. Simultaneously, PacI and I-SceI restriction sites were integrated into the ITR sequence. PCR products were detected via 1% agarose gel electrophoresis and purified using the Gel Extraction Kit (TIANquick Midi Purification Kit, Beijing, China).
We used the Gibson DNA assembly method to create fowl adenovirus vectors. Using the NEBuildER HiFi DNA Assembly Master Mix (Cat. E2623S, New England Biolabs, Ipswich, MA, USA), 200 ng of pShuttle-FAdVITR-FR plasmid backbone and 1 µg of FAdV-4 genome were mixed, and the mixtures were incubated at 50 • C for 60 min in a thermocycler. One microliter of the assembled product was used to transform EZ10 competent cells. The cells were spread on kanamycin-containing LB agar plates and incubated at 37 • C overnight.
The positive colonies were picked and grown in liquid LB medium containing kanamycin at 37 • C overnight with shaking. Colony PCR was performed with the primers FAdV4-23500-F/FAdV4-24501-R and FAdV4-ITR-F/FAdV4-1176-R (Table A1). The products were detected on a 1% agarose gel, and the plasmids were extracted. The plasmids carrying the FAdV-4 genome were named pShuttle-FAdV-4. The plasmids of consistent size in colonies were digested with BamHI and PacI, visualized on a 1% agarose gel, extracted, and confirmed to contain the viral genome.

Rescue of FAdV-4 Infectious Clones
To rescue FAdV-4 infectious clones, 80% confluent monolayer of LMH cells in 6-well plates were transfected overnight with Lipofectamine 3000 (Thermo Fisher Scientific-CN, Shanghai, China) with 5 µg of the PacI-linearized adenovirus plasmid pShuttle-FAdV-4 including genome (recovered via ethanol precipitation). Then, the medium was changed to DMEM plus 2% FBS. When the CPE reached 80%, the culture was harvested. The culture was freeze-thawed for three rounds and centrifuged at 15,292× g for 10 min to remove cell debris. The suspensions were named rFAdV-4 (P0 generation). The viral titer was detected via TCID 50 , and the viruses were serially passaged in LMH cell culture until a full cytopathic effect occurred. The recombinant adenoviruses were passaged for 3 generations (P3).

Construction of Replicating Single-Cycle FAdV-4 Vector
Homologous recombination was used to knock out the pIIIa gene with positive and negative screening markers. The thermal-sensitive pKD46 plasmid with λ-red homologous recombinase was transformed through electroporation into competent pShuttle-FAdV-4/DB3.1 cells. After being evenly distributed on LB agar plates containing kanamycin, the cells were cultured for one day at 30 • C. The pKD46 and pShuttle-FAdV-4/DB3.1 positive colonies were selected and cultivated in liquid LB medium with kanamycin and ampicillin at 30 • C with shaking at 220 r/min. Cell concentration was determined by measuring absorption at OD 600nm . L-arabinose was added to induce the expression of the homologous recombination system encoded by the plasmid pKD46.
The pKD46 and pShuttle-FAdV-4/DB3.1 competent cells were transformed together with the donor of the entire ccdB cm expression cassette via electroporation. The cells were spread on LB agar plates containing kanamycin, ampicillin, and chloramphenicol and incubated at 30 • C overnight.
The plasmids were further linearized via PmeI digestion, and ccdB cm was isolated from agarose gel, self-ligated, and transformed into EZ10 competent cells for screening to obtain pShuttle-FAdV-4(∆IIIa:PmeI)/EZ10, after which the IIIa gene was deleted. The deletion of IIIa gene-positive colonies was validated by colony PCR with IIIa-up-300-F/IIIadown-200-R primers.

Virus Isolation and Identification
The adenovirus was isolated and identified through the inoculation of SPF chicken embryo with homogenized suspected tissues, and the chicken embryos were then inoculated into LMH cells. Normal LMH cells showed plum-blossom-like growth ( Figure 1a). CPE of LMH cells was observed post-transfection during incubation at 37 • C in a humidified atmosphere supplemented with 5% CO 2 . At 72 h, typical CPE was observed ( Figure 1b). Using viral culture supernatant DNA as templates, PCR amplification yielded a specific fragment of the hexon gene ( Figure 1c). The amplification product of the hexon gene was recovered and cloned into the T vector pMD19-T, which was successfully constructed by sequencing verification. The hexon sequence was aligned with fowl adenovirus reference sequences from the GenBank database using ClustalW and classified according to standard taxonomic classifications. A phylogenetic tree was constructed using the neighbor-joining method, using the p-distance method (on 1000 bootstrapped datasets), revealing that the wild strain (Shaanxi2018) belonged to the serotype FAdV-4 ( Figure 1d). The isolate was located in the same branch as some of the FAdV-4 reference strains, which showed higher homology and closer affinity.
ified atmosphere supplemented with 5% CO2. At 72 h, typical CPE was observed ( Figure  1b). Using viral culture supernatant DNA as templates, PCR amplification yielded a spe cific fragment of the hexon gene ( Figure 1c). The amplification product of the hexon gene was recovered and cloned into the T vector pMD19-T, which was successfully constructed by sequencing verification. The hexon sequence was aligned with fowl adenovirus refer ence sequences from the GenBank database using ClustalW and classified according to standard taxonomic classifications. A phylogenetic tree was constructed using the neigh bor-joining method, using the p-distance method (on 1000 bootstrapped datasets), reveal ing that the wild strain (Shaanxi2018) belonged to the serotype FAdV-4 ( Figure 1d). The isolate was located in the same branch as some of the FAdV-4 reference strains, which showed higher homology and closer affinity.   analysis. The tree was constructed using MEGA 7.0 software according to the neighbor-joining method (1000 replicates for bootstrap). represents the isolate in this study.

Animal Regression Test
At 24 h after 10 chicks were intramuscularly injected with the P3-generation virus, huddling, chills, and depression were observed, and food intake decreased. After 72 h, two chicks died, with a 20% mortality rate, which corresponded to the clinical symptoms and histopathology of natural infections. No histopathological lesions were observed in the negative control group (Figure 2a). However, acute necrotic hepatitis with hemorrhagic, friable liver, hemorrhagic, swollen and yellowish (Figure 2b), and pericardial effusion, in which a clear straw-colored effusion is visible in the pericardial sac (Figure 2c).
represents the isolate in this study.

Animal Regression Test
At 24 h after 10 chicks were intramuscularly injected with the P3-generation virus, huddling, chills, and depression were observed, and food intake decreased. After 72 h, two chicks died, with a 20% mortality rate, which corresponded to the clinical symptoms analysis. The tree was constructed using MEGA 7.0 software according to the neighbor-joining method (1000 replicates for bootstrap). represents the isolate in this study.

Animal Regression Test
At 24 h after 10 chicks were intramuscularly injected with the P3-generation virus, huddling, chills, and depression were observed, and food intake decreased. After 72 h, two chicks died, with a 20% mortality rate, which corresponded to the clinical symptoms and histopathology of natural infections. No histopathological lesions were observed in the negative control group (Figure 2a). However, acute necrotic hepatitis with hemorrhagic, friable liver, hemorrhagic, swollen and yellowish (Figure 2b), and pericardial effusion, in which a clear straw-colored effusion is visible in the pericardial sac (Figure 2c).

Construction of FAdV-4 Infectious Clones
The pShuttle-FAdV-4 plasmid was created according to the schematic diagram shown below (Figure 3a). First, the plasmid backbone pShuttle-FAdVITR-FR was amplified by pShuttle-PacI-ITR-F/pShuttle-PacI-ITR-R primers. The amplified products were detected in a 1% agarose gel, revealing an approximately 3 kb fragment (Figure 3b). The FAdV-4 genome was then assembled into the pShuttle-FAdVITR-FR plasmid backbone. Two pairs of primers (FAV4-23500-F/FAV4-24501-R and FAV4-ITR-F/FAV4-1176-R) were used for colony PCR to determine the size of amplified fragments. PCR products were analyzed on a 1% agarose gel electrophoresis, showing approximately fragments of 1000 bp and 1200 bp, respectively (Figure 3c); this indicated that the products likely contained the viral genome. Positive bacterial colonies were determined via electrophoresis of the extracted plasmid, which shows the predicted length ( Figure 3d). These plasmids were further digested with BamHI to determine whether these plasmids bearing adenovirus genomes were generally constructed using a λ-red recombinase system, and they were confirmed to contain the viral genome via restriction analysis ( Figure 3e); positive colonies were sequenced via sequence alignment and no mutations were found. These plasmids were linearized with PacI, then the FAdV-4 genome and pShuttle-FAVITR-FR vector were observed via agarose gel electrophoresis (Figure 3f). Finally, we successfully constructed the vectors containing FAdV-4 genome via Gibson Assembly.

Construction of FAdV-4 Infectious Clones
The pShuttle-FAdV-4 plasmid was created according to the schematic diagram shown below (Figure 3a). First, the plasmid backbone pShuttle-FAdVITR-FR was amplified by pShuttle-PacI-ITR-F/pShuttle-PacI-ITR-R primers. The amplified products were detected in a 1% agarose gel, revealing an approximately 3 kb fragment (Figure 3b). The FAdV-4 genome was then assembled into the pShuttle-FAdVITR-FR plasmid backbone. Two pairs of primers (FAV4-23500-F/FAV4-24501-R and FAV4-ITR-F/FAV4-1176-R) were used for colony PCR to determine the size of amplified fragments. PCR products were analyzed on a 1% agarose gel electrophoresis, showing approximately fragments of 1000 bp and 1200 bp, respectively ( Figure 3c); this indicated that the products likely contained the viral genome. Positive bacterial colonies were determined via electrophoresis of the extracted plasmid, which shows the predicted length ( Figure 3d). These plasmids were further digested with BamHI to determine whether these plasmids bearing adenovirus genomes were generally constructed using a λ-red recombinase system, and they were confirmed to contain the viral genome via restriction analysis ( Figure 3e); positive colonies were sequenced via sequence alignment and no mutations were found. These plasmids were linearized with PacI, then the FAdV-4 genome and pShuttle-FAVITR-FR vector were observed via agarose gel electrophoresis (Figure 3f). Finally, we successfully constructed the vectors containing FAdV-4 genome via Gibson Assembly.

Rescue of FAdV-4 Infectious Clones
The virus was rescued from PacI-linearized pShuttle-FAdV-4 transfected LMH cells, as shown in the following diagram (Figure 4a). Fifteen of the eighteen plasmids bearing the Ad genome produced an infectious adenovirus clone and demonstrated a visible CPE and proliferative capacity similar to that of wild-type FAdV-4. The negative control group and partial clones CPE are as follows (Figure 4b): the supernatant in 10 days post-transfection was collected and the virus titers (P0), determined by measuring TCID 50 , ranged from 10 7 to 10 8 TCID 50 (Table A2).

Rescue of FAdV-4 Infectious Clones
The virus was rescued from PacI-linearized pShuttle-FAdV-4 transfected LMH cells, as shown in the following diagram (Figure 4a). Fifteen of the eighteen plasmids bearing the Ad genome produced an infectious adenovirus clone and demonstrated a visible CPE and proliferative capacity similar to that of wild-type FAdV-4. The negative control group and partial clones CPE are as follows (Figure 4b): the supernatant in 10 days post-transfection was collected and the virus titers (P0), determined by measuring TCID50, ranged from 10 7 to 10 8 TCID50 (Table A2).

Rescue of FAdV-4 Infectious Clones
The virus was rescued from PacI-linearized pShuttle-FAdV-4 transfected LMH cells, as shown in the following diagram (Figure 4a). Fifteen of the eighteen plasmids bearing the Ad genome produced an infectious adenovirus clone and demonstrated a visible CPE and proliferative capacity similar to that of wild-type FAdV-4. The negative control group and partial clones CPE are as follows (Figure 4b): the supernatant in 10 days post-transfection was collected and the virus titers (P0), determined by measuring TCID50, ranged from 10 7 to 10 8 TCID50 (Table A2).

Generation of a Replicating Single-Cycle Adenovirus Vector
To construct a replicating single-cycle adenovirus vector, we showed a schematic diagram of how to delete the IIIa gene (Figure 5a). pKD46 was transformed into pShuttle-FAdV-4/DB3.1 competent cells to utilize λ-red homologous recombinase to obtain pKD46 and pShuttle-FAdV-4/DB3.1 cells. These were further transformed with the donor of the whole ccdB cm expression cassette via electroporation to delete the IIIa gene in the FAdV-4 genome. The cells were spread on four agar plates containing kanamycin, ampicillin, and chloramphenicol and incubated at 30 • C overnight. Three hundred colonies grew on each plate, two of which were picked and grown in liquid LB medium containing kanamycin, ampicillin, and chloramphenicol at 30 • C with shaking. Colony PCR was performed with IIIa-up-300-F/IIIa-down-200-R primers, the size of the pShuttle-FAdV4/DB3.1 vector band in the negative control was approximately 2300 bp, and a positive fragment (2000 bp) was observed due to replacement of the IIIa gene with the ccdB cm expression cassette (Figure 5b). The plasmids obtained above were digested by the PmeI enzyme, and the target bands of about 1500 bp were digested, which were the same size as the bands of the ccdB cm expression cassette (Figure 5c). These results indicate that the colonies were carrying recombinant Ad vectors in which the IIIa gene was deleted and replaced by the corresponding ccdB cm expression cassette. The pKD46 plasmid could replicate normally at 30 • C, but the thermosensitive replicators could not replicate at 42 • C and could be eliminated by the culture at 42 • C, and the elimination of pKD46 plasmid was verified by checking whether ampicillin resistance existed. The pKD46 eliminated strain was named pShuttle-FAdV-4(∆IIIa:ccdB-Cm)/DB3.1. To cure the ccdB cm expression cassette, the pShuttle-FAdV-4(∆pIIIa:ccdB-Cm) plasmid was further linearized by digestion with PmeI and used to transform EZ10 competent cells to verify ccdB cm expression cassette deletion. Colony PCR was performed with the primers of IIIa-up-300-F/IIIa-down-200-R, the size of the pShuttle-FAdV-4(∆pIIIa:ccdB-Cm) vector band in the negative control was 2000 bp, and a positive fragment (500 bp) in pShuttle-FAdV-4(∆IIIa:PmeI) vector was observed due to deletion of the ccdB cm gene (Figure 5d). The ccdB cm expression cassette was successfully removed.

Generation of a Replicating Single-Cycle Adenovirus Vector
To construct a replicating single-cycle adenovirus vector, we showed a schematic diagram of how to delete the IIIa gene (Figure 5a). pKD46 was transformed into pShuttle-FAdV-4/DB3.1 competent cells to utilize λ-red homologous recombinase to obtain pKD46 and pShuttle-FAdV-4/DB3.1 cells. These were further transformed with the donor of the whole ccdB cm expression cassette via electroporation to delete the IIIa gene in the FAdV-4 genome. The cells were spread on four agar plates containing kanamycin, ampicillin, and chloramphenicol and incubated at 30 °C overnight. Three hundred colonies grew on each plate, two of which were picked and grown in liquid LB medium containing kanamycin, ampicillin, and chloramphenicol at 30 °C with shaking. Colony PCR was performed with IIIa-up-300-F/IIIa-down-200-R primers, the size of the pShuttle-FAdV4/DB3.1 vector band in the negative control was approximately 2300 bp, and a positive fragment (2000 bp) was observed due to replacement of the IIIa gene with the ccdB cm expression cassette (Figure 5b). The plasmids obtained above were digested by the PmeI enzyme, and the target bands of about 1500 bp were digested, which were the same size as the bands of the ccdB cm expression cassette (Figure 5c). These results indicate that the colonies were carrying recombinant Ad vectors in which the IIIa gene was deleted and replaced by the corresponding ccdB cm expression cassette. The pKD46 plasmid could replicate normally at 30 °C, but the thermosensitive replicators could not replicate at 42 °C and could be eliminated by the culture at 42 °C, and the elimination of pKD46 plasmid was verified by checking whether ampicillin resistance existed. The pKD46 eliminated strain was named pShuttle-FAdV-4(ΔIIIa:ccdB-Cm)/DB3.1. To cure the ccdB cm expression cassette, the pShuttle-FAdV-4(ΔpIIIa:ccdB-Cm) plasmid was further linearized by digestion with PmeI and used to transform EZ10 competent cells to verify ccdB cm expression cassette deletion. Colony PCR was performed with the primers of IIIa-up-300-F/IIIa-down-200-R, the size of the pShuttle-FAdV-4(ΔpIIIa:ccdB-Cm) vector band in the negative control was 2000 bp, and a positive fragment (500 bp) in pShuttle-FAdV-4(ΔIIIa:PmeI) vector was observed due to deletion of the ccdB cm gene (Figure 5d). The ccdB cm expression cassette was successfully removed.

Discussion
The major capsid proteins of FAdV-4 include hexon, penton, and fiber, which make up the majority of the virion surface. The hexon protein contains specific antigenic determinants for adenovirus population, type, and subtype identification, and is currently widely used for virus isolation and identification [38,39]. We isolated a fowl adenovirus that was Viruses 2023, 15, 1657 9 of 12 identified by a phylogenetic tree based on the hexon gene sequence. The fowl adenovirus isolation was classified as serotype FAdV-4 in this study. The virus isolation targets the liver and heart as the main target organs and can cause typical clinical symptoms and pathological changes in animal regression tests. Infected chicks showed 20% mortality, indicating strong virulence of the virus isolation.
Considering the size of the viral genome, the efficiency and convenience of constructing infectious clones remains a challenge. Initially, adenovirus infectious clone construction involves the homologous recombination of a linearized shuttle plasmid carrying a homologous arm with DNA from the adenovirus-containing full-length genome in mammalian cells and screening for plaques containing recombinant adenovirus. This method has major drawbacks due to its low efficiency in homologous recombination and the need for multiple rounds of time-consuming and laborious screening and identification. Later, it was determined that the recA enzyme carried by E. coli itself or the introduction of the λ-red recombinase system in E. coli can achieve DNA recombination between shuttle plasmids and adenovirus full-length genomes in E. coli. Further optimized and improved Gibson Assembly technology can insert approximately 300 kb of target fragments into the target vector. In addition, the SLiCE method and in-fusion technology can also enable recombination between fragments of interest in vitro. In this study, the FAdV-4 vector was initially constructed using in-fusion technology, but the connection efficiency was low. Meanwhile, also in this study, the FAdV-4 vector was successfully constructed via Gibson Assembly, and infectious clones were successfully rescued in LMH cells. The Gibson Assembly method, which is widely used in the construction of infectious clones [40][41][42], has the advantages of convenient operation, simplicity, repeatability, and high efficiency, especially in the construction of adenovirus infectious clones, which has broad application prospects. In addition, TG1 strain was initially used for homologous recombination in this study, but the recombination efficiency was very low and there were few positive clones. Considering the instability of TG1 strain, EZ10 strain was subsequently changed to be more stable for recombination, and a large number of positive clones were obtained. Therefore, when conducting homologous recombination, the selection of more stable recombinant strains would greatly improve the positive efficiency.
Infectious clones can modify or mutate the viral genome, available for fundamental research, gene therapy, and the production of recombinant vaccines [23,25,43,44]. The SC-Ad was created primarily by deleting cement protein IIIa should be taken into consideration as they can replicate the genome and transgenes as well as RC-Ad but do not produce infectious progeny viruses [34,35,45]. Therefore, the safety profile of SC-Ad suggests that these vectors may be useful for vaccines and vector amplification for other therapies. In this study, an RC-Ad vector based on the infectious clone of FAdV-4 was constructed using the λ-red recombineering system. Currently, the world's leading gene knockout systems include Cre-Loxp, CRISPR/Cas9, and λ-red in vivo homologous recombination technology. We also established the optimal design of paired sgRNAs targeting the IIIa to induce on-target sequence mutation but did not effectively elicit CRISPR/Cas9-mediated cleavage, possibly due to FadV-4 genome insertion in the high-copy vector backbone with the pBR332 ori.
The deficiency of this study is that the deletion of the IIIa gene based on infectious clones requires further virus packaging by complementing the IIIa gene through the cell line. This will be addressed in future work. In the future, we will also study the SC-Ad vector of the FAdV-4 as a vaccine platform.

Conclusions
In this study, a strain was isolated from a sick flock and identified as FAdV-4 through hexon gene sequencing phylogenetic analysis. Subsequently, infectious clones of FAdV-4 were successfully generated using Gibson Assembly technology and rescued by transfecting LMH cells. In addition, the IIIa gene was deleted based on infectious clones to construct the SC-Ad vector of FAdV-4. Institutional Review Board Statement: All animal experiments were performed according to the guidelines of the Northwest A&F University, using protocols approved by the institutional laboratory of the Animal Care and Use Committee (NO. 220413). The 3R principle was strictly observed during the experiment to ensure animal welfare.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data available on request from the authors.

Acknowledgments:
We extend our sincere appreciation to the laboratory personnel for their technical proficiency and invaluable assistance in conducting animal experiments. The content has been approved by all authors who have made significant contributions to the work.

Conflicts of Interest:
The authors declare no conflict of interest. Table A1. Primers used in the study.  Table A2. Virus titer determination.