Localization and distribution of goose astrovirus 2 antigens in different tissues at different times

Goose astrovirus 2 (GAstV-2) causes visceral gout in goslings and has resulted in significant economic losses in the goose industry of China since its outbreak in 2017. To further investigate the distribution and localization of GAstV-2 in different tissues at different times, a monoclonal antibody (mAb)-based immunohistochemical (IHC) assay was developed to detect GAstV-2. A total of 80 1-day-old healthy goslings were inoculated with GAstV-2 via the oral (n = 40) and intramuscular routes (n = 40). GAstV-2 in the tissues of interest was detected using the established IHC assay. The results showed that positive signals were detected in most tissues at 1 day post-infection (dpi). Viral antigens were mainly distributed in the cytoplasm, and the staining intensity was higher in the renal tubular epithelial cells than in other cells. Taken together, our data demonstrated that GAstV-2 has a broad tissue tropism and primarily targets the kidneys. These results are likely to provide a scientific basis for further elucidation of the pathogenesis of GAstV-2.


Introduction
Astroviruses (AstVs) are single-stranded, positive-sense and non-enveloped RNA viruses with a whole genome length of 6.4-7.9 kb that belong to the Astroviridae family [1].The AstV genome consists of a 5′-untranslated region (UTR), three open reading frames (ORF1a, sensitive and specific serological diagnostic method that can be used for immune evaluation and serosurveillance of GAstV-2 infection.However, qRT-PCR, LAMP and ICS are mainly used for the testing of clinically infected samples, while ic-ELISA is primarily utilized for clinical serological diagnosis and immune evaluation.Unlike the above methods, immunohistochemistry (IHC) is an immunological detection method that can qualitatively and quantitatively measure the antigen of interest via antigen-antibody and histochemical reactions.This method not only directly reveals the level of damage caused by the pathogen to the body, but it also unveils the location of the pathogen in various organs, which can provide a basis for understanding the site and mechanism of action of the pathogen [12].At present, IHC has been extensively used for disease diagnosis and pathogen detection [13][14][15].
For this reason, we developed a reliable IHC assay to detect GAstV-2 antigens in different tissues at different time points after infection in the hopes of further elucidating the pathogenesis of GAstV-2.

Virus
In 2021, a strain of GAstV-2 named GAstV-SDTZ (Gen-Bank accession number: OP221731) was isolated from goslings suffering from visceral gout (Zaozhuang City, Shandong Province), and was used as the challenge virus for experimental infection.The infectivity titer of the virus was determined to be 10 − 4.9 TCID 50 /0.1 mL by infection of the chicken liver cell line (ATCC) using the Reed-Muench assay [16].The challenge virus was free of other waterfowl-dervied viruses (including GAstV-1, H9N2 subtype avian influenza virus, avian orthoreovirus, Tembusu, goose parvovirus, Newcastle disease virus and fowl adenovirus).

Animal infection
One-day-old healthy goslings were purchased from a commercial hatchery in Jining city, Shandong province, China [17].A total of 120 one-day-old healthy goslings were divided into three groups (A, B, and C) with 40 goslings per group.The goslings were reared in different negative pressured isolators.Goslings were inoculated with 0.3 mL of viral suspension (GAstV-SDTZ strain; 10 − 4.9 TCID 50 /0.1 mL) orally in group A and intramuscularly in group B (infected groups).Goslings in group C were orally inoculated with 0.3 mL sterile PBS (control group).Clinical signs and mortality were monitored and recorded daily.

Histopathology
Tissue samples (e.g., liver and kidneys) were collected from each group for histopathology.Tissues were fixed in 10% neutral formalin at room temperature for 48 h, processed and embedded in paraffin, cut into 5 μm paraffin sections, stained with hematoxylin and eosin (H&E), and observed under a light microscope (Nikon, EclipseE100, Japan).

Immunohistochemistry
Tissue samples were collected from each group for IHC analysis.The tissue sections were dewaxed and then treated with anhydrous ethanol for 3 min, 95% ethanol for 3 min, 85% ethanol for 3 min, 75% ethanol for 3 min and deionized water for 3 min.Antigen retrieval was performed using sodium citrate (pH 6.0) for 7 min under microwave heating, and the tissue sections were then blocked with 5% bovine serum albumin (Beyotime Biotechnology Shanghai, China) for 30 min at 37 °C.Tissue sections were incubated with the primary monoclonal antibody (mAb; 1:1000, prepared by our laboratory as described by Yang et al.) for 12 h at 4 °C [10], incubated with biotinylated goat anti-mouse secondary IgG (1:500; CWBIO, Beijing, China) for 1 h at 37 °C, stained with DAB (Beyotime Biotechnology Shanghai, China) for 5 min, stained with hematoxylin for 2 min, and examined under a light microscope (Nikon, EclipseE100, Japan).

Specificity of IHC assay
The positive control was the tissue of dead goslings infected with GAstV-2, and the negative control was the tissue of healthy goslings in the control group.The primary antibody was replaced with PBS in the blank test.Mouse anti-goose Tembusu mAb was used as an isotype control for mouse anti-GAstV-2 mAb.The specificity of the IHC assay was determined using liver sections of goslings infected with GAstV-1, H9N2 subtype avian influenza virus, avian orthoreovirus, goose parvovirus, Tembusu, Newcastle disease virus and fowl adenovirus.

Statistical analysis
Data were analyzed using one-way analysis of variance in GraphPad Prism 8.0 (GraphPad Software Inc.).

Clinical signs and gross lesions
In groups A and B, loss of appetite and depression were observed at 3 to 5 dpi.A total of 13 goslings in group A died between 4 and 13 dpi, and 11 goslings in group B died between 5 and 12 dpi (Fig. 1).At necropsy, the kidneys of both infected groups showed severe haemorrhage and swellings.
Necropsy revealed significant pathological changes in the kidneys of both infected groups, including severe hemorrhages and renal enlargement.Moreover, gross lesions including urate deposits on the surface of the visceral organs, such as the liver, heart, and kidneys, were observed in dead goslings (Fig. 2).All goslings in group C were healthy during the experimental period.

Histopathology of different tissues
Histopathological changes were predominantly found in the kidneys and liver.Renal tubular epithelial cell degeneration, necrosis and exfoliation were observed in the kidneys, whereas steatosis, vacuolar degeneration and inflammatory cell infiltration were detected in the liver (Fig. 3A-D).In addition, no microscopic histological lesions were observed in the control group.

Specificity of the IHC assay
Our results showed that the IHC assay established in this study was only positive for the positive control, and negative for all other control tests.

Localization and distribution of GAstV-2 in goslings
The liver, spleen, lungs, kidneys, bursa of Fabricius, thymus, pancreas, brain, glandular stomach, and duodenum stained positive for GAstV-2 in dead goslings infected with the virus, confirming the broad tissue tropism of GAstV-2 (Fig. 4).Positive signals could be detected in the liver, spleen, lungs, kidneys, thymus, pancreas, glandular stomach, and duodenum at 1 dpi in both infected groups.Positive signals could be detected in the bursa of Fabricius at 3 dpi and in the brain at 5 dpi.Though, positive signal was no longer detectable in the bursa of Fabricius, pancreas, and brain at 21 dpi in both infected groups (Table 1).In addition, positive signals were detected in the glandular stomach and duodenum at 1 dpi in group B and at 3 dpi in group A. Staining intensity was significantly higher from 7 to 12 dpi than on other days, with the highest in the kidneys (Table 2).

Discussion
GAstV-2 associated goose gout has caused huge economic damage to the goose industry of China [6].Currently, qRT-PCR and LAMP are used for the detection of Previous studies have shown that viral nucleic acid can be detected in all investigated tissues of infected goslings [18].We examined the distribution of the viral antigens in goslings at different times points after GAstV-2 infection using the established IHC assay and found that positive signals were widely present in various cell types, including hepatocytes, lymphocytes surrounding the central splenic artery, squamous and cubic epithelial cells of the bronchus, epithelial cells of proximal convoluted tubules, distal convoluted tubules and collecting tubules of the kidney, reticular structure in the medulla of the thymus lobule, cytoplasm of B cells and D cells in pancreatic islets, neurons, columnar epithelial cells of the glandular gastric mucosa, duodenal and intestinal gland columnar cells, and Paneth cells.This suggests that GAstV-2 has a broad tissue tropism, which is consistent with other recent reports.
The staining intensity was similar between the oral and intramuscular inoculation groups, and was significantly higher in the kidneys than in other tissues, indicating that the kidneys have the highest viral load, followed by the liver.The bursa of Fabricius showed a positive signal at 3 dpi, and the brain showed a positive signal at 7 dpi.However, viral nucleic acid could be detected in the bursa of Fabricius and brain at 2 dpi by qRT-PCR, which may be attributed to the lower sensitivity of the IHC assay for viral antigen during the early stage of infection when the tissue viral load is low.In addition, positive signals were detected earlier in the glandular stomach and duodenum in the oral inoculation group than in the intramuscular inoculation group, which may be associated with the route of infection and the metabolic pathways induced by the virus.Though, further studies are warranted to confirm these findings.
Uric acid is the end product of purine metabolism [19] and is excreted predominantly by multiple urate transporters on renal proximal epithelial cells [20].Our results revealed that the staining intensity was significantly higher in the proximal convoluted tubules, distal convoluted tubules and collecting tubules of the kidneys than in other tissues, indicating that renal tubular epithelial cells are the main target cells of GAstV-2 and injury of renal proximal epithelial cells may be an important cause of obstructed uric acid excretion and gout.
Taken together, this study demonstrated that GAstV-2 antigen is widely distributed across various tissues at

Fig. 2 Fig. 1
Fig. 2 Gross lesions of goslings infected with GAstV-SDTZ.(A) Urate deposits on the surface of the liver and heart; (B-C) Severe hemorrhage and swelling in the kidneys; (D-F) Uninfected goslings

Fig. 4
Fig. 4 IHC detection of viral antigens in different tissues of goslings after GAstV-SDTZ infection.(A-A1) Cytoplasm of hepatocytes, branches of hepatic portal vein and hepatic artery; (B-B1) Lymphocytes around the central artery; (C-C1) Flat epithelial cells and cuboidal epithelial cells lining the bronchi, respiratory capillary (D-D1) Cytoplasm of epithelial cells of proximal convoluted tubules, distal convoluted tubules and collecting ducts; (E-E1) Medulla of bursa of Fabricius; (F-F1) Cytoplasm of reticular cells in the medulla; (G-G1) Cytoplasm of B cells and D cells in pancreatic islets; (H-H1) Cytoplasm of a neuron; (I-I1) Cytoplasm of glandular cells in the compound duct gland and cytoplasm of columnar epithelial cells in the glandular gastric mucosa; (J-J1) Cytoplasm of columnar cells in the duodenal gland, Paneth cell and smooth muscle cells of mucous membrane; (a-j) Uninfected goslings