Genetic characterization and phylogenetic analysis
Amplification of a 698 bps fragment spanning part of the NP and the F2 gene of NDV was performed using SuperScript III One-Step RT-PCR system (Invitrogen, Carlsbad, CA, USA) following published protocols (Aldous et al., 2003). Amplicons were size-separated by agarose electrophoresis, excised and purified from gel using the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). Purified PCR products were used directly for cycle sequencing reactions using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequencing reaction products were purified using NucleoSeq kit (Macherey-Nagel, Düren, Germany) and sequenced on an ABI PRISM® 3100 Genetic Analyzer (Life Technologies, California, USA).
Assembly of the obtained sequences was performed using the Geneious software, version 9.0.5 (Kearse et al., 2012). Alignment and identity matrix analyses were conducted using MAFFT (Katoh & Standley, 2013) and BioEdit (Hall, 1999). Sequences generated in this study were submitted to the GenBank NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/) and assigned accession numbers shown in Table 1. Reference sequences of NDV genotypes (Diel et al., 2012; Snoeck et al., 2013) required for further analyses were retrieved from the GenBank database. Phylogenetic analyses were performed and trees were constructed using the IQ-tree software version 1.1.3 (Minh et al., 2013; Nguyen et al., 2014) based on maximum likelihood analysis of phylogenetic relationship after selection of the best fit substitution model. Finally, trees were viewed and edited using FigTree v1.4.2 software (http://tree.bio.ed.ac.uk/software/figtree/) and Inkscape 0.51.