2.3 Virus strain and artificial infection test
The strain N-DRV-XT18 (GenBank Accession No. MK749398-MK749407) used in this study was a variant duck orthoreovirus strain isolated from the spleen of Pekin duck with splenic swelling and necrosis isolated in 2018. LMH (Leghorn Male-chicken Hepatocellular-carcinoma, ATCC CRL-2013) cell was used for virus isolation and proliferation, according to conventional virus cell culture methods (Tang and Lu, 2015a). The median tissue culture infective dose (TCID50) of the N-DRV-XT18 strain was 10−6.52/0.1mL, which was calculated using the Reed-Muench assay. Before the inoculation, the virus culture supernatant was tested for purity by PCR to exclude other pathogenic contamination (Wang et al., 2020). (The sequence of the primers was not shown).
60 healthy female and 15 male breeding ducks (35-weak-old) were purchased from a breeding farm in Tai’an city, Shandong province. Before the experiment, each duck collected serum samples and cloacal swabs, and tested by ELISA and qPCR to confirm that its N-DRV antibody test was negative, and it did not contain N-DRV infection or other duck pathogens. These ducks were randomly divided into three groups, orally administered group, intramuscular injection group and control group, each group of 20 female ducks and 5 male ducks. The orally administered and intramuscular injection group were inoculated with 0.2mL (10-6.52/0.1mL) virus culture supernatant. The control group was inoculated with equal doses of stroke-physiological saline solution (SPSS) at the same injection site (Wang et al., 2020). The three groups were reared in separate houses and ensured that the feeding conditions were consistent and free from contamination by external pathogens.