Detection and Sequencing of Avian Avulaviruses

Samples were subjected to RNA extraction from the allantoic fluid using an RNA extraction kit (RNAeasy Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Detection of avian avulaviruses was conducted using a real-time reverse transcriptase PCR based on the M gene of NDV and the N gene of aMPV as described previously [30 (link)]. For full length fusion (F) gene amplification, RNA was reverse transcribed into cDNA using a Superscript IV First-Strand cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA) and the second strand was synthesized with Q5 DNA Polymerase (New England Biolabs, Ipswich, MA, USA) using for amplification and sequencing of the full length F gene [31 (link),32 (link)]. Amplified PCR products were visualized by electrophoresis on a 1.2% agarose gel electrophoresis and then purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The purified PCR products were sequenced bi-directionally with both sense and antisense primers that were used in the PCR amplification [31 (link),32 (link)] using ABI PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA) by Sanger sequencing method on a 3500 Applied Biosystems capillary sequencer (Source Bioscience, Cambridge, UK).