Isolation and characterization of an astrovirus causing fatal visceral gout in domestic goslings

Zhang, Qingshui; Cao, Yanxin; Wang, Jun; Fu, Guanghua; Sun, Mengxu; Zhang, Lijiao; Meng, Li; Cui, Guolin; Huang, Yu; Hu, Xueying; Su, Jingliang

Virulent bacteria were not isolated and tissue samples were negative by PCR for goose parvovirus, goose hemorrhagic polyomavirus, reovirus, or Tembusu virus. However, a DNA fragment was amplified from the RNA sample extracted from the pooled spleen, liver and kidney tissues using pan-AstV RT-PCR targeting the AstV RNAdependent RNA polymerase (RdRp) gene 21 . Sequence and phylogenetic analysis of the amplified RdRp gene with other known AstV sequences retrieved in the GenBank database showed that the detected virus could be assigned to the subgroup 1.2 within the avastrovius group 1, with the closest relationship to the astrovirus detected from dropping samples of northern shovelers (Anas clypeata) in Hong Kong (Fig. 2) 22 . However, the nucleotide sequence of the RdRp gene had ≤67.5% similarity to the sequences of other astroviruses within avastrovirus group 1, suggesting that the virus was genetically distinct from known avastroviruses. Therefore, the isolation of AstV was initiated by inoculating tissue samples into goose embryos. For the first inoculation, significant thickening of the embryo's chorioallantoic membrane was noted although no death occurred by 5 days post inoculation (dpi). The subsequent passage of the isolate caused 60-100% mortality of the embryos by 5 dpi. The dead embryos exhibited severe subcutaneous edema and hemorrhages with necrotic foci in the liver (Fig. 3 ). Using the gene specific RT-PCR, the AstV was consistently detected in the allantoic fluids. Quantal assays showed that the infectious virus titers of the embryo allantoic fluid increased from 5 × 10 4 ELD 50 /ml for the fourth passage to 5 × 10 5.5 ELD 50 /ml for the 9 th passage, indicating that the isolates adapted to the goose embryo culture system. Therefore, the isolate was designated AAstV/Goose/ CHN/2017/SD01 (SD01 hereafter) as proposed by Martella et al. 23 .