RNA extraction and RT-PCR
Total RNA was extracted from liver filtrate by MiniBEST Universal RNA Extraction Kit (TaKaRa,Dalian, China), following the manufacturer’s instructions. The extracted RNA was carried out using One-step RT-PCR Kit (TaKaRa, Dalian, China) with specific primers (DAstV-F 5’-CTTGGACTGTGGAAGCATATACC-3’, DAstV-R 5’-GTTGAAAACTGCCCTGAAGG-3’) to amplify a partial sequence of approximately 763 bp of a DAstV-1, and PCR amplicons were sequenced for confirmation.
Virus isolation and sequencing
For virus isolation a selection of liver sample (ZaoZhuang County) from Shandong province that was tested positive for DAstV-1 by RT-PCR was prepared. 0.5 ml of the filtered suspension was inoculated into LMH cells (ATCC CRL-2013), a chicken hepatocellular carcinoma cell line. The cultures incubated at 37℃with 5% CO2 and examined for a cytopathic effect (CPE) daily. The cells and supernatant within 4-7 days post inoculation (PI) were snap-frozen for further passages.
Next-generation sequencing
The RNA extracted from the DAstV-SDZZ sample was subjected to complete genome sequencing by next-generation sequencing (NGS) with the Hiseq platform following the manufacturer’s instructions. The NGS was performed at the Shanghai Personal Biotechnology Co., Ltd, China. The details of the NGS library preparation are the same as we described earlier (Wei et al., 2020). Specific primers were developed to verify the genomic sequence of the isolate as described previously (R. Zhang et al., 2019).