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).