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.