Newcastle disease is one of the most serious diseases of birds, caused by Newcastle disease virus (NDV), also known as avian paramyxovirus type 1 (APMV-1). The disease is distributed world-wide and may cause large economic losses in the poultry industry. The severity of the disease varies depending on the host species and specific properties of the virus. Highly virulent strains of NDV can be discriminated from low virulent strains by the presence of a multibasic amino acid motif at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) are low virulent for chickens, despite their F protein cleavage site containing a multibasic amino acid sequence. Furthermore, these isolates may show an increase in virulence when passaged in chickens. To study the molecular basis of NDV virulence, a reverse genetics system for the non-virulent PPMV-1 strain AV324 was developed. Furthermore, the already available reverse genetics system for the highly virulent NDV strain Herts/33 was used. Subsequently, the NP, P, M, F and L genes were evaluated for their role in virulence by exchanging these genes between both strains. The results showed that the F protein of AV324 is functionally not different from that of Herts/33. However, a direct correlation between the activity of the viral replication proteins (NP, P and L) and virulence was demonstrated. To elucidate the adaptation mechanism by which PPMV-1 becomes more virulent for chickens, strain AV324 was passaged five times in day-old chickens. Complete genome sequencing of passaged virus showed that the increase in virulence was accompanied by mutations in the L and P replication proteins rather than the viral fusion protein. The effect of these mutations on virulence and enhanced replication was confirmed by means of reverse genetics. All together, these results demonstrate that the virulence level of NDV is directly related to the efficiency of viral replication. These data, together with the knowledge that some PPMV-1 infected pigeons shed virus without having clinical signs and the observation that the number of NDV outbreaks in pigeons caused by PPMV-1 in Europe is increasing, underlines the potential danger of pigeon-derived paramyxoviruses for the poultry industry
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