Figure 3

Detection of infective ASLV viral particles in amniotic fluid from SPF chicken embryos. (A-D) Inoculation assay for detection of exogenous ASLV infection. DF-1 chicken fibroblast cells cultured in the presence of (A) no amniotic fluid; (B) amniotic fluid from untreated SPF chicken embryo; and (C) amniotic fluid from SPF chicken embryo infected with RCAS-nucGFP construct. Inoculated cells were processed for immunohistochemical detection of ASLV viral protein p27 (red) and counterstained with DAPI (blue). Lack of immunohistochemical signal in cells inoculated with amniotic fluid from SPF untreated embryos (B) suggested that SPF embryos were not pre-infected with exogenous ASLV virus. (A) and (C) were used as negative and positive controls respectively. af: amniotic fluid. Scale bar:20 μm. (D). RT-PCR analysis of supernatants from (-) non-inoculated DF-1 cells; or DF-1 cells inoculated with (+) amniotic fluid from SPF embryos infected with RCAS-nucGPF; or (1-3) untreated SPF embryos, confirmed absence of exogenous viral infection in SPF embryos. (E) Detection of endogenous infective viral particles directly from amniotic fluid. Amniotic fluid from (+) SPF embryos infected with RCAS-nucGPF; or (1) untreated SPF embryos negative for p19/p27 by IHC; or (2-3) untreated SPF embryos positive for p19/p27 by IHC, was used for direct RT-PCR detection of infective ASLV viral particles. Positive RT-PCR amplification from amniotic fluid from embryos purposely infected with RCAS-nucGFP (+) set proof-of-principle for the approach. On the other hand, lack of RT-PCR amplification in amniotic fluid from SPF untreated embryos further demonstrated absence of both, exogenous and endogenous ASLV infective viral particles. (-) RT-PCR negative control.