Several studies have been done using molecular techniques to determine the F0 cleavage site sequence by
reverse-transcription polymerase chain reaction (RT-PCR), either on the isolated virus or on tissues and
faeces from infected birds, followed by analysis of the product by restriction enzyme analysis, probe
hybridisation or nucleotide sequencing with a view to establishing a routine in vitro test for virulence (Miller et
al., 2010b). Determination of the F0 cleavage sequence may give a clear indication of the virulence of the
virus, and this has been incorporated into the definition of ND