Virulent APMV-1 strains contain a multibasic cleavage site (with the general consensus sequence of RRQKRQF) with a polybasic furin motif (RX[R/K]RQ) that is readily cleaved in cell culture by intracellular furin or furin-like protease. Avirulent APMV-1 strains lack this polybasic site and depend on extracellular protease for cleavage, which in vitro can be supplied by exogenous protease and in vivo is supplied by secretory trypsinlike protease found in the lumen of the respiratory and enteric tracts [20] . Our study showed that APMV-1 strain BC readily forms syncytia in cell culture and replicates more efficiently in vitro than any of the other APMVs, followed by the APMV-1 LaSota strain. Characterization of the CPE of APMV types 2-9 demonstrated that only APMV-3 and -5 induced syncytium formation in infected cells. The cleavage site of APMV-3 (RPRGRQL) lacks the furin motif, and efficient growth and syncytium formation depended on supplementation with exogenous protease, resembling an avirulent APMV-1 strain. The cleavage site of APMV-5 (KRKKRQF) is the only example from APMV2-9 to have a furin motif [6] , and syncytium formation and growth were independent of added protease, resembling a virulent APMV-1 strain. Production of syncytia by APMV-3 and APMV-5 correlated with efficient multicycle replication in cell culture in the case of APMV-3 and a moderate level of replication in cell culture in the case of APMV-5. Although APMV-5 contains the greatest number of basic residues for any of the APMV except for some strains of APMV-1, its replication in DF1 cells was much less efficient compared to APMV-1 and -3. Interestingly, the level of replication of APMV-5 in cell culture was very similar to that of APMV-2, which lacks a furin motif (KPASRQF), does not form Figure 6 . Replication of APMVs in 1-day-and 2-week-old chickens. Groups of (A) 1-day-or (B) 2-week-old chickens were inoculated with each virus (256 HA units) by the intranasal route. Three birds from each group were sacrificed on 3 dpi (1-day-old chicks) or 4 dpi (2-week-old chickens), and virus titers in the collected tissues samples (brain, trachea, lung, and spleen) were determined by limiting dilution in DF1 cells and immunostaining with polyclonal antibodies raised against the respective N protein. doi:10.1371/journal.pone.0034927.g006 syncytia, and whose replication and lack of ability to form syncytia are independent of exogenous protease. Thus, for those two viruses (APMV-2 and -5), the growth phenotype in cell culture did not correlate well with the presence or absence of a furin motif in the cleavage sequence. The other APMVs (types 4, 6, 7, 8, and 9) have one or two basic amino acids in their cleavage site sequences and exhibited single cell infection and inefficient replication in vitro. However, only in the case of serotypes 8 and 9 did the addition of exogenous protease increase the efficiency of replication, and the overall level of replication remained very low and syncytia were not observed. Thus, APMV types 2, 4, 6, 7, 8, and 9 have cleavage sites that seemed similar to those of avirulent APMV-1 strains, but the inclusion of protease had little effect on growth in cell culture.