Analysis of Newcastle disease virus quasispecies and factors affecting the emergence of virulent virus.

Genome sequence analysis of a number of avirulent field isolates of Newcastle disease virus revealed the presence of viruses (within their quasispecies) that contained virulent F0 sequences. Detection of these virulent sequences below the ~1% level, using standard cloning and sequence analysis, proved difficult, and thus a more sensitive reverse-transcription real-time PCR procedure was developed to detect both virulent and avirulent NDV F0 sequences. Reverse-transcription real-time PCR analysis of the quasispecies of a number of Newcastle disease virus field isolates, revealed variable ratios (approximately 1:4-1:4,000) of virulent to avirulent viral F0 sequences. Since the ratios of these sequences generally remained constant in the quasispecies population during replication, factors that could affect the balance of virulent to avirulent sequences during viral infection of birds were investigated. It was shown both in vitro and in vivo that virulent virus present in the quasispecies did not emerge from the "avirulent background" unless a direct selection pressure was placed on the quasispecies, either by growth conditions or by transient immunosuppression. The effect of a prior infection of the host by infectious bronchitis virus or infectious bursal disease virus on the subsequent emergence of virulent Newcastle disease virus was examined.