To evaluate neurotropism of APMVs in vitro, primary chicken neuronal cells were prepared from 9-day-old chicken embryos for virus infection. Briefly, the section of hippocampus was dissected from embryos, digested with trypsin, passed through a cell strainer (40 mm nylon), seeded onto poly-L-lysine-coated plates, and then infected with each virus [32] . Spread of viruses in neuronal cells was determined by confocal microscopy analysis. Briefly, at 48 hpi, the cells were fixed, permeabilized, stained with polyclonal antibodies against the respective N protein and a neuronal marker (anti-neuron specific beta III tubulin antibody, abcamH, Cambridge, MA) followed by anti-Alexa Fluor 488 and 594 antibodies, and then analyzed by confocal microscopy. In addition, virus replication in neuronal cells was determined by collecting supernatants at 12-h intervals until 72 hpi. Virus titers in the collected supernatants were quantified in DF1 or Vero cells by limiting dilution and immunostaining with N-specific antibodies as described above.