Role of intergenic sequences in newcastle disease virus RNA transcription and pathogenesis.

Newcastle disease virus (NDV), a member of the family Paramyxoviridae, has a nonsegmented negative-sense RNA genome consisting of six genes (3'-NP-P-M-F-HN-L-5'). The first three 3'-end intergenic sequences (IGSs) are single nucleotides (nt), whereas the F-HN and HN-L IGSs are 31 and 47 nt, respectively. To investigate the role of IGS length in NDV transcription and pathogenesis, we recovered viable viruses containing deletions or additions in the IGSs between the F and HN and the HN and L genes. The IGS of F-HN was modified to contain an additional 96 nt or more or a deletion of 30 nt. Similarly, the IGS of HN-L was modified to contain an additional 96 nt or more or a deletion of 42 nt. The level of transcription of each mRNA species (NP, F, HN, and L) was examined by Northern blot analysis. Our results showed that NDV can tolerate an IGS length of at least 365 nt. The extended lengths of IGSs down-regulated the transcription of the downstream gene and suggested that 31 nt in the F-HN IGS and 47 nt in the HN-L IGS are required for efficient transcription of the downstream gene. The effect of IGS length on pathogenicity of mutant viruses was evaluated in embryonated chicken eggs, 1-day-old chicks, and 6-week-old chickens. Our results showed that all IGS mutants were attenuated in chickens. The level of attenuation increased as the length of the IGS increased. Interestingly, decreased IGS length also attenuated the viruses. These findings can have significant applications in NDV vaccine development.