RT-qPCR, virus isolation and purification
Molecular pathotyping based on RT-qPCR using the primers and probes
specific for avirulent and virulent pathotypes (RT-F-qPCR) revealed that
two Egyptian NDV isolates of the year 2011 were positive for lentogenic
as well as virulent pathotypes, indicating mixed infection. The
consensus sequence of the proteolytic cleavage site within the
F2-protein of one mixed isolate (R1954-11) represents a
polybasic recognition motive, characteristic for virulent
meso-/velogenic pathotypes (. This would be in agreement with the
obtained ICPI of 0.91, representing a mesogenic pathotype. In contrast,
the sequence of the second mixed isolate R1973/11 represents a cleavage
site of lentogenic vaccine type viruses but had an ICPI of 1.88, clearly
identifying the isolate as velogenic. By plaque cloning, clones with
mono- (R1954/-11- cl1; R1973/11-cl 2) or polybasic cleavage sites
(R1954/11-cl 6; R1973/11-cl 4) could be obtained from both samples. For
these purified clones, unambiguous reactivity patterns in
pathotype-specific RT-qPCRs were consistent with their sequences of the
proteolytic cleavage site and their ICPI values even though the ICPI
value of R1973-11-cl 2 was slightly above the threshold of 0.7 between
lentogenic to mesogenic strains.