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.