2.2 │ virus isolation and identification
Blood samples were collected aseptically from the Hy-Line brown layer chickens, and semens were collected as sterile as possible. The semen samples were diluted 5 times with PBS containing 2% penicillin and streptomycin. After mixing upside down gently, the supernatant was centrifuged to precipitate cells at 4000 r/min for 2 min. Albumens were collected aseptically, and diluted 4 times with DMEM in 5ml syringes. Virus isolation of plasmas, semen and albumens were performed by inoculation of DF1 cells as previously described (Meng et al., 2018). The ALV group-specific antigen p27 in the culture supernatant was detected by an enzyme-linked immunosorbent assay.
The cell wells were washed with PBS and fixed with cold acetone–alcohol mixture (3:2) for 5 min. Then, the cells were incubated with mouse anti-ALV-J monoclonal antibody JE9 (Qin et al., 2001) at 37°C for 60 min, following by incubation with goat anti-mouse IgG antibody conjugated with fluorescein isothiocyanate (Sigma, California, USA) at 37°C for another 60 min. Finally, the cells were observed under fluorescence microscope.