Clinical evaluation

The clinical scoring was performed in accordance with the evaluation grids provided by the local ethical commission. Mice were assessed daily for weight loss and clinical symptoms. In particular, different parameters were monitored: activity, posture, gait, coat, type of respiration, state of activity, and deshydratation. Excessive loss of body weight (>25%) or obvious sign of suffering were considered as limit points in the experiments.

ELISA, quantitative PCR, and measurement of PVM viral loads Quantification of MuHV-4 specific antibodies by ELISA and quantification of MuHV-4 genomic and cellular DNA copies by quantitative PCR were performed as described.⁵⁸ PVM genomes from lung tissue were measured by quantitative RT-PCR with dual standard curve as previously described.¹⁶ Infectious PVM loads at euthanasia were quantified by plaque assay from co-culture of lung homogenate dilutions with BHK-21 permissive cells.