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.