The Isolation and Molecular Characterization of an Astrovirus From “Yellow” Chickens, China

Figure 1. Isolation and characterization of CAstV NJ1701 isolate in LMH cell culture. After four passages, the virus was isolated and identified in LMH cells. (A) Uninfected LMH cells at 48 h post infection (p.i.) (NC, upper panel) and LMH cells infected with NJ1701 at 48 h p.i. (lower panel), which are detached with small and round cells present in the media (400×). (B) The viral gene copy numbers in cell supernatant were 1.13 × 104 and 1.21 × 106 copies/μl at 24 and 48 h p.i., respectively. Data were expressed as mean ± SD from three independent experiments. ***P < 0.001. (C) Indirect immunofluorescence assay was performed at 24 and 48 h post-infection with mouse-anti-CAstV serum. Immunofluorescence results of CAstV show cytoplasmic staining in the infected LMH cells.

Figure 2. Predicted genome organization of CAstV NJ1701. Three predicated ORFs with their locations for CAstV NJ1701 are shown. The translation start sites of ORF1a and ORF2 are indicated by black triangles. The nucleotide position of the start site of the heptameric AAAAAAC (RFS) sequence are shown by gray triangles. Black bars are the untranslated regions and the 24-nt spacer between the stop and start codons of ORF1b and ORF2, respectively. ORF, open reading frame; RdRp, RNA-dependent RNA polymerase; RFS, ribosomal frameshift signal; s2m, stem-loop-II-motif.

Figure 3. Phylogenetic relationships between CAstVs in this study and other avian astroviruses. The phylogenetic analysis of chicken astrovirus isolate NJ1701 was based on the nucleotide sequences of the complete genome (A) and on amino acid sequences of the complete ORF1a (B), ORF1b (C), and ORF2 (D) regions using neighbor-joining method with 1,000 bootstraps.

Figure 4. Infection experiments with CAstV NJ1701 in chicken embryos. (A) Viral gene copy numbers of allantoic fluid in embryos of different passages. The copy numbers of passages 1, 2, and 3 were 6.00 × 103, 4.58 × 104, and 1.59 × 105, respectively. Data were expressed as mean ± SD from three independent experiments. (B) Ten days after infection at passage 3, 17-day-old infected embryos with growth depression (two of the left) were observed. The uninfected embryo (right) was used as control. (C) The viral copy numbers of different visceral organs from chicks at hatch in infected group were determined using qRT-PCR. Data were expressed as mean ± SD from three independent experiments.