Molecular characterization of chicken astrovirus and pathogenicity of a novel isolate in China

Figure 1. Clinical cases (n = 31) of CAstV infections in 1 day-old chicken flocks between 2020 and 2022 in China. (A) Geographical distribution of clinical cases of CAstV. (B) Temporal distribution of clinical cases of CAstV. (C) The presence of other viral pathogens.

Figure 2. Virus isolation and identification of the CAstV strain JS202103 in LMH cells. (A) LMH cells control. (B) CPE formation in LMH cells infected with the JS202103 showing as detached small and round at 48 h post-infection (black arrow). (C,D) LMH cells were fixed at 48 h post-infection, and incubated with CAstV-specific mouse antisera followed by incubation with Cy3-conjugated goat anti-mouse secondary antibody (red fluorescence). Then, the cells were nuclear stained with DAPI (blue fluorescence).

Figure 3. Phylogenetic tree of ORF2 amino acid sequences of 31 isolated strains (black solid triangles) and 57 reference strains using neighbor-joining method of the MEGA 7 0.0 software program with 1,000 bootstrap replicates. CAstV strains which belong to subgroup Ai are labelled with purple branches, CAstV strains which belong to subgroup Aii are labelled with dark-yellow branches, CAstV strains which belong to subgroup Aiii are labelled with dark-green branches, CAstV strains which belong to subgroup Bi are labelled with light-green branches, CAstV strains which belong to subgroup Bii are labelled with orange branches, CAstV strains which belong to subgroup Biii are labelled with red branches, CAstV strains which belong to subgroup Biv are labelled with light-blue branches, CAstV strains which belong to subgroup Bv are labelled with dark-blue branches, CAstV strains which belong to subgroup Bvi are labelled with pink branches.

Figure 4. Phylogenetic analyses performed on the basis of the nucleotide sequences of full-length genomes (A), amino acid sequences of ORF1a (B), and amino acid sequences of ORF1b (C) of the CAstV strain JS202103 with the neighbor-joining method in MEGA 7 0.0 software program (1,000 bootstrap replicate). A black solid triangle was used to label new isolate.

Figure 5. Macroscopic lesions from JS202103-inoculated and negative control 17 days-old chicken embryos. (A–C) No gross lesions were found in control chicken embryos. (D) The presence of green colour in the allantoic fluid. (E) The heart is covered with extensive white urate deposits (black arrow), and white thin membranous covering of the liver (white arrow). (F) Congested and enlarged kidneys with deposition of urate crystals (white arrow), and intestines filled with yellow and greenish content (black arrow).

Figure 6. Haematoxylin and eosin-stained tissue sections from JS202103-inoculated and negative control 17 days-old chicken embryos. (A–D) Microscopic picture of a control chicken embryo. (E–H) Microscopic picture of a CAstV-infected chicken embryo. (F) Mild vacuolar degeneration of hepatocytes, small vacuoles in the cytoplasm (black arrow), mild congestion of hepatic sinuses (red arrow), and some brownish-yellow substance deposition (yellow arrow) were observed in the liver. (G) Extensive destruction of renal tubules, detachment of tubular epithelial cells, karyopyknosis and deep staining (black arrow), unclear glomerular capillary plexus as well as decreased cells (red arrow) in the kidney. (H) Extensive shedding of intestinal mucosa epithelium and exposed intestinal lamina propria were found in the intestine.

Figure 7. Viral load in allantoic fluids and various tissue samples of CAstV-infected SPF chicken embryos (n = five embryos).