Full Genome Sequencing
Full genome sequences of four successfully isolated NDV strains, namely, R1954-11-cl-1, R1973-11-cl-2, R1973-11-cl-4 and AR2178-14, representing three genotypes previously reported in Egypt, were carried out. Briefly, RNA was purified from isolates using TRIzol LS reagent (LifeTechnologies, Darmstadt, Germany) and an RNeasy mini kit (Qiagen, Hilden, Germany) with on-column DNase digestion according to the manufacturer’s instructions. Conversion of RNA into double-stranded DNA was performed using a cDNA synthesis system (Roche, Mannheim, Germany). Library construction was done as previously described (Juozapaitis et al., 2014). Sequencing was performed on an Illumina MiSeq Instrument using the MiSeq reagent kit version 3 (Illumina, San Diego, CA, USA).
Assembly of the sequence data was done using the Genome Sequencer version 2.6 software suite (Roche), and NDV-related contigs were identified with BLASTn (BLASTn; http://blast.ncbi.nlm.nih.gov/Blast.cgi). The obtained full-length NDV sequences have been submitted to GenBank (Table 1). Further, the open reading frame (ORF) analysis and the annotation of the genome were carried out using the Geneious software.
Phylogenetic analyses were carried out for both of F-gene (partial/full) and the whole genome sequence separately using references viruses as mentioned in the previous section.