Viral RNA Extraction and Influenza A Subtyping

Viral RNA was extracted from the collected samples using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. All samples were initially tested using standard quantitative reverse transcription polymerase chain reaction (RT-qPCR) specific for the M gene of influenza A viruses [25 (link)]. Positive RNAs were further tested using gene-specific RT-qPCR assays for the hemagglutinin (HA) and neuraminidase (NA) gene segments of the AIV H5N8 subtype [26 (link)]. Viral RNAs were further tested for the presence of infectious bronchitis virus (IBV) [27 (link)] and Newcastle disease virus (NDV) [28 (link)]. All RT-qPCR reactions were performed using Stratagene MX3005P real-time PCR machine (Agilent, Santa Clara, CA, USA). Virus isolation was performed through allantoic fluid inoculation of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECEs) according to the standard protocols of the OIE diagnostic manual [29 ]. SPF ECEs were obtained from the Nile SPF project (Koom Oshiem, Fayoum, Egypt). The harvested allantoic fluids were tested for virus hemagglutination activity by hemagglutinin assay and verified by using RT-qPCR.