Figure 3

Cytopathogenicity of AV and WT reovirus on embryonic stem cells. (A) Pluripotent murine ESCs (R1 and D3) were infected with WT or AV reovirus (MOI of 20). At 48 hpi, cell lysates were prepared and viral proteins (λ, μ and σ) were detected by immunoblotting using reovirus antiserum (left panel). Alternatively, cells were fixed/permeablized for FACS analysis at 48 h post-infection and reovirus antiserum and secondary FITC antiserum were used to detect the presence of reovirus proteins (right panel). (B) At 48 hpi, viral cytopathic effects were also photographed. (C) Severe combined immunodeficiency mice received a single implantation of murine R1 (left panel) or MES1 (right panel) ESCs. At 12 days following implantation, teratomas were intratumourally injected with WT or AV reoviruses (10⁷ PFU per mouse; n=3–5) or D Reo (Dead, UV-inactivated reovirus) or PBS and teratoma growth was monitored for 21–70 days post-infection. Mice treated with WT reovirus were killed at 18–19 days post-infection because of the formation of viral-induced myocarditis. (D) Photographs of representative R1 teratomas were taken 21 days after implantation (9 days post-infection).