Ethics statement
All animal infection experiments were conducted in accordance with the “Guidelines for Experimental Animals” of the Ministry of Science and Technology (Beijing, China) and were conducted under the supervision of the Animal Protection and Utilization Committee of Shandong Agricultural University.
Clinical samples
The disease started in 1 to 3-week-old ducks on rearing fields with mortality rates ranging from 10 to 30%. Their clinical signs included lethargy, loss of appetite and weight loss. Postmortem examination revealed a steatosis and haemorrhagic lesions of the liver, as well as hemorrhage and swellings in kidneys. From May to September 2020, a total of one hundred and seventy liver samples were taken from the diseased ducks from different commercial duck farms located at four provinces of China (Figure1). The tissues from different commercial duck farms were homogenized in phosphate buffered saline (PBS) (20%, w/v) containing antibiotics (100 U/ml of Penicillin and 100 μg /ml of Streptomycin), and centrifuged at 12,000×g at 4 °C for 15 minutes. The supernatant was filtered through a 0.22-μm syringe-driven filter to remove large particles and stored at −80°C until use. The existence of other avian pathogenic viruses (including AIV, TMUV, DHAV-1, DHAV-3, FAV, DPV, GPV, GoAstV and DucV) were ruled out by reverse transcription PCR (RT–PCR) or PCR.