The chicken liver hepatocellular carcinoma cell line LMH was purchased from the American Type Culture Collection (ATCC); LMH cells were cultured in DMEM/F12 (Gibco, NY, United States) supplemented with 10% fetal bovine serum (FBS) (Gibco). The FAdV-4 strain HN and FAdV-8a strain AH720 were isolated as described previously (Wang et al., 2019; Lv et al., 2021) and allowed to replicate in LMH cells. SPF chickens were purchased from Merial Vital Laboratory Animal Technologies Co., Ltd. (Beijing, China). All animal experiments were performed with strict adherence to the guidelines for animal use with approval from Shanghai Laboratory Animal Management Committee and the Animal Care and Use Committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (permit number: SYXK 2020-0027).

The pathogenicity of FAdV-4 and FAdV-8a during co-infection with these strains was demonstrated in the SPF chickens. First, 52 3-week-old SPF chickens were randomly divided into four groups (n = 13 per group). The chickens in group I and group II were challenged intramuscularly with 100 μl of 10⁵ TCID₅₀ of strain HN and 100 μl of 10⁵ TCID₅₀ of strain AH720, respectively. The chickens in group III were challenged intramuscularly with a mixture of 100 μl of 10⁵ TCID₅₀ of strain HN and 100 μl of 10⁵ TCID₅₀ of strain AH720. The chickens in group IV were intramuscularly challenged with 100 μl of PBS. At 3 days post-challenge, three chickens from each group were sacrificed. Tissue samples, including tissues from the liver, pancreas, kidney, spleen, lung, duodenum, jejunum, rectum, and cecum, were collected. These samples were divided into two parts. One part was used for DNA extraction to monitor the viral loads and the second part was fixed in 10% neutral formalin. The remaining 10 chickens from each group were monitored daily and scored for clinical signs for 14 days, as described previously (Zhao et al., 2015). The scoring scheme was as follows: 0 for normal, 1 for mild depression, 2 for severely depressed, 3 for paralysis/prostration, and 4 for death. The survival of the remaining chickens was monitored.

The viral loads in the tissues of infected chickens were determined using TaqMan probe fluorescence quantitative polymerase chain reaction (qPCR). The total DNA was isolated from the liver, pancreatic, kidney, spleen, lung, duodenal, jejunal, rectal, and cecal tissue samples. The FAdV-4 hexon gene (1293–1417 nt) was used as an indicator for the presence of HN strain DNA and the FAdV-8a fiber gene (836–904 nt) was used as an indicator for the presence of AH720 strain DNA, as described in previous studies (Wang et al., 2019; Lv et al., 2021). qRT-PCR was performed on an Applied Biosystem 7500 Fast instrument with the following cycling conditions: 95°C (5 min), 40 cycles at 95°C (10 s) and 60°C (15 s) and 60°C (30 s). The standard curves were generated, based on which the quantity of the viral DNAs in the tissue samples were calculated.

The liver, kidney, lung, and spleen tissue samples collected from three chickens in each group were fixed in 10% neutral-buffered formalin for histopathological examination. The samples were routinely dehydrated, embedded in paraffin wax, and then sectioned for hematoxylin and eosin (H&E) staining. The tissue samples were examined under a Nikon microscope equipped with an Olympus DP25 camera. The histopathological lesions were assessed using the following scoring scheme: 0 for no lesions, 1–3 for mild lesions, 4–6 for moderate lesions, and 7–10 for severe lesions (Zhao et al., 2015).

An immunofluorescence assay (IFA) was performed to investigate the distribution of the viral particles in the livers of chicken infected with the mixture of the HN and AH720 strains. The slides of livers from chickens infected with the HN and AH720 strains were serially cut, blocked using 2% BSA for 1 h, and incubated overnight at 4°C with FAdV-4 Hexon 1B4 monoclonal antibody (1:1000 dilution) and FAdV-8a Fiber polysera (1:1000 dilution) (both prepared by our lab), respectively. Following three washes in PBS buffer, the slides were incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody and Alexa Fluor 594-conjugated goat anti-mouse IgG, respectively. Whole-slide images were captured using the Pannoramic confocal 3D HISTECH system and analyzed by PanoramaStudio Pro software.

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay was performed to evaluate the degree of apoptosis in liver samples from the chickens in the different infection groups, according to the manufacturer’s instructions. The samples were incubated with 50 μl of TUNEL reaction mixture (TdT and fluorescein – dUTP) at 37°C for 60 min in a humid atmosphere. The TUNEL staining intensity was examined, and images were captured using a Pannoramic confocal 3D HISTECH system and analyzed using the PanoramaStudio Pro software. The TUNEL-positive cells were counted, and the average numbers of these cells were compared.

The data were presented as the means ± SEM. All the data were analyzed using the Prism 7 software (GraphPad, La Jolla, CA, United States). A paired two-tailed Student’s t-test was performed to compare the means of data from two groups. The differences were considered statistically significant at p-values < 0.01 or < 0.05.

To determine the pathogenicity of chickens co-infected with HN and AH720 strains, the chickens were first randomly divided into four groups and then challenged with the HN strain (group I), AH720 strain (group II), mixture of the HN and AH720 strains (group III), and PBS (group IV). During the infection period, the clinical scores varied for the chickens from the different infection groups. The clinical scores for AH720 infection were less than those for HN infection and those for HN + AH720 infection (Figure 1A). The chickens from groups I and III showed typical symptoms of HHS and IBH, with enlarged yellow and hemorrhagic liver and pericardial effusion (Figure 1B). The chickens from group II showed mild symptoms with minor liver hemorrhage (Figure 1B). The control chickens (those from group IV) showed no clinical symptoms.

Further, the mortality rates of the chickens from the different groups were investigated. The mortality rates of the chicken from groups I and III were both 100%, while no death was observed in the cases of the chickens from groups II and IV (Figure 1C). Notably, the mortality rate of the chicken from group I reached 100% within 4 days post-challenge. However, the mortality rate of the chickens from group III reached 100% at 6 days post-challenge (Figure 1C). These data suggested that co-infection with the HN and AH720 strains was slightly less lethal to the chickens than the infection with the HN strain alone.

To investigate the distribution and viral loads of the HN and AH720 strains in infected chickens, viral copy numbers in different tissues from the FAdV-infected chickens were determined at 3 days post-challenge using the previously established TaqMan probe RT-PCR for FAdV-4 and FAdV-8a (Wang et al., 2019; Lv et al., 2021), respectively.

In chickens from group I, the HN strain was detected in all the tissue samples, with the highest counts in the liver (approximately 2.5 × 10⁷ copies/mg), followed by the pancreas, jejunum, kidney, duodenum, and cecum (Figure 2). In the chickens from group II, the AH720 strain was detected in most tissue samples, but the viral loads were relatively low, with the highest viral load being observed in the cecum (approximately 1 × 10⁴ copies/mg) (Figure 2). However, the viral loads of the HN and AH720 strains in the chickens from group III differed markedly from these in the chickens from group I and group II. On one hand, the viral loads of the HN strain in the liver, pancreas, jejunum, kidney, and duodenum in the chickens from group III were significantly lower than those in the corresponding organs of chickens from group I. On the other hand, the viral loads of the HN strain in the cecum, rectum, spleen, and lungs from the chickens in group III were higher than those in the corresponding organs from the chickens in group I (Figure 2). In addition to the pancreas and jejunum, a significant increase in the viral load of the AH720 strain was observed in all the tested tissue samples from the chickens in group III, compared with that in the tested tissue sections from the chickens in group II (Figure 2). These results suggested that the interaction between the HN and AH720 strains may influence the viral replication and proliferation of the HN and AH720 strains in different tissues.

At 3 days post-challenge, the liver, kidney, lung, and spleen tissues from the chickens in the four different groups were fixed for histopathological analysis. Pathological lesions were observed in various tissues of chickens in the different infection groups (Figure 3). The liver tissues from the chickens in group I showed severe liver lesions and presented typical basophilic inclusions with many infiltrating lymphocytes (Figure 3A1). Hepatocyte necrosis was found in the liver tissues of the chickens from group II (Figure 3A2). Lymphocyte infiltration and hepatocyte necrosis were observed in the liver tissues of the chickens from group III (Figure 3A3). No histological changes were observed in the livers of the chickens from the control group (Figure 3A4).

Renal tubular structural disorder was observed in the kidney tissues from chickens in all the three infection groups (Figures 3B1–B3). Inflammatory exudation was observed in the kidneys of the chickens from groups I and III (Figures 3B1,B3). Severe renal hemorrhage was seen in the kidneys of the chickens from group I (Figure 3B1). No histological changes were observed in the kidneys of the chickens from the control group (Figure 3B4).

Structural disorder of the pulmonary bronchus, inflammatory exudation, and alveolar rupture were observed in the lung tissues from the chickens in group I (Figure 3C1). The lung tissues from the chickens in group II were relatively normal (Figure 3C2). Desquamation of pulmonary epithelial cells was observed in the lung tissues from the chickens in group III (Figure 3C3). No significant histological changes were observed in the lung tissues from the chickens in the control group (Figure 3C4).

The number of lymphocytes was reduced in the spleen tissues from the chickens in group I (Figure 3D1). Splenic hemorrhage was observed in the spleen tissues from the chickens in group II (Figure 3D2). Splenic hemorrhage and reduced lymphocytes were observed in the spleen tissues from the chickens in group III (Figure 3D3). No significant histological changes were observed in the spleen tissues from the chickens in the control group (Figure 3D4).

Compared with the control groups, the histopathological lesions in the liver, kidney, lung, and spleen tissues from the chickens in the infection groups were notable (Figure 4). Among the four sampled tissues, the liver tissue showed the highest histopathological scores; the highest histopathological scores were observed for the livers from the chickens in group I (Figure 4).

The liver tissues collected from the chickens in group III were serially sectioned to investigate the distribution of the viruses in the livers of chickens co-infected with the HN and AH720 strains by IFA. The presence of the HN strain was seen as a green color that was detected by incubation with FAdV-4 Hexon antibody, and subsequently, incubation with Alexa Fluor 488-conjugated secondary antibody. The presence of the AH720 strain was seen as a red color that was detected by incubation with FAdV-8a Fiber antibody, and subsequently, incubation with Alexa Fluor 594-conjugated secondary antibody. IFA analysis of the liver slides revealed that in the chickens co-infected with the HN and AH720 strains, the HN strain presented a scattered distribution and the AH720 virus particles were accumulated in the hepatic cells (Figure 5). This result corresponded with that observed from the histopathological examination of the liver tissues from the chickens co-infected with the HN and AH720 strains.

To further investigate the apoptosis of the liver cells in the chickens from the different infection groups, the collected liver tissue samples were analyzed by TUNEL staining. As shown in Figure 6, the number of TUNEL-positive cells in the livers of chickens from group I was the highest, but this number was not statistically significant compared with that in the livers of chickens from group III. However, the number of TUNEL-positive cells in the livers of chickens from group II was significantly less than that in the livers of chickens from groups I and III. These data indicated that the decrease of the viral load of the HN strain was compensated by the increase of the viral load of the AH720 strain in the liver samples from the chickens in group III, resulting in a similar apoptotic level being observed in the samples from the chickens in group I.