FAdV-4 strain SD2015 and FAdV-8b strain JSSQ15 were isolated and maintained in our laboratory (Ye et al., 2016). FAdV-7 and leghorn male hepatoma (LMH) cells were purchased from ATCC. FAdV-8a strain AH720 was kindly provided by Prof. Hongjun Chen (Shanghai Veterinary Research Institute). The LMH cells were cultured in F12/DMEM (Gibco, NY, USA) with 10% fetal bovine serum (FBS) (Lonsera, Shanghai, China).

Plasmids pcDNA3.1-Fiber, pcDNA3.1-Hexon, and pcDNA.1-Penton (fiber, hexon, and penton from FAdV-8b) were constructed and kept in our laboratory. Plasmids pcDNA3.1-F1, pcDNA3.1-F1-Tail-Flag, pcDNA.1-F1-Shaft-Flag, pcDNA3.1-F1-Knob-Flag, pcDNA3.1-F1-Tail+Shaft-Flag, and pcDNA3.1-F1-Shaft+Knob-Flag (F1 was the Fiber-1 of FAdV-4) were stored in our laboratory (Wang et al., 2020).

His-Fiber-1 of FAdV-4 and His-Fiber-1 of duck adenovirus 3 (DAdV-3) were generated by the expression in E. coli BL2, purified by Ni column, identified by SDS-PAGE, and preserved in our laboratory. The monoclonal antibody (mAb) 1B5 against the hexon of FAdVs was provided by Prof. Hongjun Chen (Shanghai Veterinary Research Institute). The mAb 4D9 against fiber of FAdV-8b, positive chicken sera against FAdV-8b, the mAb 3B5 against Fiber-1 of FAdV-4, and anti-chicken CAR polyclonal antibodies were generated and stored in our laboratory. Anti-Flag mouse mAb (Cat F1804) was purchased from Sigma-Aldrich Co. LLC.

Leghorn male hepatoma cells were transfected with the different recombinant plasmids (pcDNA3.1-Fiber, hexon, and penton of FAdV-8b, pcDNA3.1-Fiber-1 of FAdV-4 and Fiber-1 truncations of FAdV-4). After 24 h, LMH cells transfected with plasmids were infected with the indicated virus at a multiplicity of infection (MOI) of 0.01. At 24, 48, 72, 96, and 120 h post-infection (hpi), the supernatants of infected cells were collected and titrated according to the 50% tissue culture infection dose (TCID₅₀), as previously described (Lu et al., 2022).

Purified His-Fiber-1 of FAdV-4 was first mixed with FAdV-8b at an MOI of 0.01 and subsequently inoculated into the LMH cells. At the same time, purified His-Fiber-1 of DAdV-3 was set as a control. At 2 hpi, the infected LMH cells were washed twice with PBS and then maintained with fresh F12-DMEM containing 1% FBS. At day 5 post-infection, the LMH cells were collected and analyzed by Western blot using mAb 1B5 against the hexon of FAdVs and mAb 4D9 against the fiber of FAdV-8b.

Leghorn male hepatoma cells transfected with the plasmids or infected with the viruses were fixed with a prechilled acetone-ethanol (3:2) mixture for 5 min and washed twice with PBS. The fixed cells were incubated with diluted antibodies for 45 min at 37°C. Then, the cells were washed thrice with PBS and incubated with the diluted secondary antibodies (fluorescein isothiocyanate (FITC)-labeled IgG) for another 45 min at 37°C. After washing with PBS thrice, the cells were observed using a fluorescence microscope.

The LMH cells were collected and lysed with lysis buffer (CoWin Biosciences, Taizhou, China) with protease and phosphatase inhibitor cocktail (New Cell and Molecular Biotech, Suzhou, China) on ice for 30 min. After high-speed centrifugation at 4°C, the lysates supernatant was boiled with protein loading buffer. After protein samples were separated by SDS-PAGE, the separated proteins were transferred onto nitrocellulose (NC) membranes. After blocking with blocking buffer (New Cell and Molecular Biotech, Suzhou, China) for 20 min at room temperature (RT), the NC membranes were incubated with the primary antibody diluted in PBST containing 5% skimmed milk at 4°C overnight. After three washes with PBST, the NC membranes were incubated with horseradish peroxidase (HRP)-labeled IgG at RT for 1 h. After another three washes with PBST, the membranes were developed on an ECL substrate and analyzed using an automatic imaging system (Tanon 5200).

The sgRNA against the chicken CAR (XM_416681.5) was designed using the website (www.benchling.com) and then cloned into the lentiCRISPR V2 plasmid. The primers used for sgRNA cloning were as follows: forward primer, 5′-CACCGTGTGTTGCTTCAAACCGAGT-3′ and reverse primer, 5′-AAACACTCGGTTTGAAGCAACACAC-3′. Then, LMH cells were first transfected with gRNA targeting chicken CAR. At 48 h post-transfection, the CAR-KO cell lines were selected using the proper concentration of puromycin, followed by limited dilution, sequencing, and Western blot analysis as previously described (Li et al., 2021).

Leghorn male hepatoma cells in 6-well plates were infected with the indicated virus at an MOI of 0.01. At 2 hpi, the inoculated cells were washed twice with PBS and then maintained with F12-DMEM containing 1% FBS. The cell supernatants collected at 24, 48, 72, 96, and 120 hpi were measured for viral replication kinetics as previously described (Lu et al., 2022).

All data are presented as the mean ± standard deviation. In addition, statistical analysis in this study was performed using a Student's t-test or a one-way ANOVA t-test using GraphPad 6 software. p < 0.05 is considered statistically significant. ^*, ^**, ^***, and ^**** indicate p < 0.05, 0.01, 0.001, and 0.0001, respectively.

To identify which protein directly mediates the infection of FAdV-8b, we performed a superinfection resistance assay by transfecting pcDNA3.1-Fiber, pcDNA3.1-Hexon, and pcDNA.1-Penton into LMH cells. Similar transfection efficacies and expression levels of fiber, hexon, and penton of FAdV-8b in the transfected LMH cells were verified by IFA (Figure 1A). The superinfection resistance assay revealed that the viral titer of FAdV-8b in the LMH cells transfected with pcDNA3.1-Fiber, pcDNA3.1-Hexon, and pcDNA3.1-Penton presented no difference to that in cells transfected with the control vector pcDNA3.1 at each time point (Figure 1B). Taken together, these data demonstrate that the fiber, hexon, and penton of FAdV-8b could not efficiently block or inhibit the viral replication of FAdV-8b in the susceptible cell line LMH.

Recently, our group and Pan et al. reported that the Fiber-1 of FAdV-4 directly mediated the infection of FAdV-4 via the cellular receptor CAR (Pan et al., 2020; Wang et al., 2020). To investigate whether FAdV-8b can infect LMH cells through a similar cell receptor used by FAdV-4, we tested the superinfection resistance assay against FAdV-8b using the Fiber-1 of FAdV-4. In this assay, FAdV-7 and FAdV-8a were used as control viruses. As shown in Figure 2A, similar expression levels of Fiber-1 in the LMH cells transfected with pcDNA3.1-F1 were confirmed in the three infection groups (FAdV-7, FAdV-8a, and FAdV-8b). The superinfection resistance assay revealed that the viral titer of FAdV-8b in the LMH cells transfected with pcDNA3.1-F1 was significantly lower than that of the LMH cells transfected with the control pcDNA3.1 at each time point (Figure 2B). Notably, the viral titer of FAdV-8b in the LMH cells transfected with pcDNA3.1-F1 was 100 times lower than that of the LMH cells transfected with the control pcDNA3.1 at 96 hpi. However, the viral titers of FAdV-7 and FAdV-8a in the cells transfected with pcDNA3.1-F1 were very similar to those of the LMH cells transfected with the control pcDNA3.1 (Figure 2B). Western blot analysis further confirmed the superinfection resistance activity of the Fiber-1 of FAdV-4 against the infection FAdV-8b. As shown in Figure 2C, the abundant hexon protein of FAdV-8b was found in the cells transfected with pcDNA3.1 at 120 hpi, whereas that in cells transfected with pcDNA3.1-F1 was barely detected. Besides, no significant difference was observed in the expression of hexon protein in FAdV-7 and FAdV-8a either transfected with pcDNA3.1-F1 or pcDNA3.1. These data demonstrate that the Fiber-1 protein of FAdV-4 can efficiently inhibit the viral replication of FAdV-8b but not FAdV-7 and FAdV-8a in LMH cells.

To confirm the superinfection resistance against FAdV-8b by Fiber-1 of FAdV-4, FAdV-8b was first mixed with the purified His-Fiber-1 of FAdV-4 and then inoculated into LMH cells. The His-Fiber-1 of DAdV-3 was used as a control. As shown in Figure 3A, the viral titer of FAdV-8b in the cells incubated with His-Fiber-1 of FAdV-4 was significantly lower than that of cells incubated with His-Fiber-1 of DAdV-3 at each time point. It should be noted that the viral titer of FAdV-8b in the cells incubated with His-Fiber-1 of FAdV-4 was approximately 100 times lower than that of cells incubated with His-Fiber-1 of DAdV-3 at 96 hpi. In addition, Western blot analysis further confirmed the interfering activity of His-Fiber-1 of FAdV-4 against the infection FAdV-8b. The hexon and fiber protein of FAdV-8b in the infected cells treated with His-Fiber-1 of FAdV-4 were significantly lower than those of cells treated with His-Fiber-1 of DAdV-3 (Figure 3B). Together, these data clearly demonstrate that the purified His-Fiber-1 of FAdV-4, but not the purified His-Fiber-1 of DAdV-3, can efficiently interfere with the replication of FAdV-8b in LMH cells.

To determine which domain of the Fiber-1 of FAdV-4 contributes to the superinfection resistance against FAdV-8b, a set of Fiber-1 truncations with different domains fused with a flag tag at the C-terminus which were designated as pcDNA3.1-F1-Tail-Flag, pcDNA3.1-F1-Shaft-Flag, pcDNA3.1-F1-Knob-Flag, pcDNA3.1-F1-Tail+Shaft-Flag, and pcDNA3.1-F1-Shaft+Knob-Flag (Fiber-1 truncations), stored in our group, were used. The transfection efficacies and expressions of these Fiber-1 truncations were confirmed by IFA using anti-Flag mAb. As shown in Figure 4A, all of these Fiber-1 truncations were efficiently expressed with similar transfection efficacies. The superinfection resistance assays using these Fiber-1 truncations showed that the viral titers of FAdV-8b in the cells transfected with pcDNA3.1-F1 or pcDNA3.1-F1-Shaft+Knob-Flag were significantly lower than those of cells transfected with pcDNA3.1 at 48, 72, and 120 hpi (Figure 4B). However, the viral titers of FAdV-8b in the cells transfected with other Fiber-1 truncations were very similar to those of cells transfected with pcDNA3.1 at each time point (Figure 4B). Moreover, western blot analysis further confirmed the superinfection resistance activity against the infection FAdV-8b, conferred by shaft and knob domains of the Fiber-1 of FAdV-4. As shown in Figure 4C, the hexon and Fiber protein of FAdV-8b in the cells transfected with pcDNA3.1-F1 or pcDNA3.1-F1-Shaft+Knob-Flag at 120 hpi were barely detected, whereas in contrast, abundant hexon and fiber protein were detected in cells transfected with pcDNA3.1 and other Fiber-1 truncations. Taken together, these data demonstrate that shaft and knob domains of FAdV-4 Fiber-1 are responsible for the superinfection resistance against FAdV-8b.

Previously, Pan et al. identified CAR homology as a cellular receptor for FAdV-4 through its binding with Fiber-1, and our group found that the shaft and knob domains of Fiber-1 mediate the infection of FAdV-4 (Pan et al., 2020; Wang et al., 2020). Hence, to investigate whether CAR plays a vital role in FAdV-8b infection, an LMH cell line with a knockout of CAR was first generated using the CRISPR/Cas9 technique, designated as CAR-KO LMH cells, and further identified by western blot (Figure 5A). As shown in Figure 5B, the viral titers of FAdV-8b in CAR-KO LMH cells were ~50 times lower than that of wild-type (WT) LMH cells at 48 hpi, although no differences were observed at other time points. Western blot analysis further confirmed that knockout of CAR in LMH cells inhibited FAdV-8b replication at 48 hpi. As shown in Figure 5C, the band indicating the FAdV-8b hexon in CAR-KO LMH cells was significantly lower than that of WT LMH cells at 48 hpi. In summary, all these data demonstrate that CAR has a significant effect on the early infection of FAdV-8b in vitro.