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

To sequence the complete genome of the isolate, total RNA was extracted from the goose embryo allantoic fluids of the fourth passage using a viral RNA kit (Omega, GA, USA) and the cDNA was synthesized using a Reverse Transcription System (Promega, WI, USA) with random primers following the manufacturer's instructions. Viral genomic fragments were amplified by PCR with primer sets designed against conserved regions of the AstV sequences retrieved from the GenBank database (Table 2) . PCR products were purified using Gel Extraction Kit (Omega, GA, USA) and ligated into pEASY-Blunt Simple Cloning Vector (TransGen Biotech, Beijing, China). The recombinant vector was transformed into competent Escherichia coli Trans-T1(TransGen Biotech, Beijing, China) and transformants containing the PCR amplified fragment were selected by PCR following the manufacturer's instruction. At least two representative transformants were subjected to bidirectional DNA sequencing using Applied Biosystems ABI3730 (Shanghai Meiji Biological Medicine Technology Co., Ltd. Shanghai, China). The 5′ and 3′ ends of the viral genome were amplified using the 5′/3′ RACE kit (Clontech, CA, USA) following the guidelines of the manufacturer. The initial complete genome was assembled and manually edited using the Software ContigExpress. Based on the initial genome sequence, additional primer pairs were designed (Table S3) , and PCR amplicons were sequenced to determine the genome.