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.