Improved Subtyping of Avian Influenza Viruses Using an RT-qPCR-Based Low Density Array: 'Riems Influenza a Typing Array', Version 2 (RITA-2)

• Published in: Viruses 2022-02, Vol.14 (2), p.415
• Main Authors: Hassan, Kareem E, Ahrens, Ann Kathrin, Ali, Ahmed, El-Kady, Magdy F, Hafez, Hafez M, Mettenleiter, Thomas C, Beer, Martin, Harder, Timm
• Format: Article
• Language: English
• Subjects: Animals; Avian flu; avian influenza; Birds - virology; Bronchitis; Diagnosis; Equidae - virology; Exo-a-sialidase; Genomes; Glycoproteins; Hemagglutinin Glycoproteins, Influenza Virus - genetics; Hemagglutinins; Humans; Infections; infectious bronchitis virus; Infectious bronchitis virus - genetics; Infectious bronchitis virus - isolation & purification; Influenza; Influenza A; Influenza A virus - classification; Influenza A virus - genetics; Influenza A virus - isolation & purification; Laboratories; Neuraminidase - genetics; Newcastle disease; Newcastle disease virus; Newcastle disease virus - genetics; Newcastle disease virus - isolation & purification; Polymerase chain reaction; Poultry; Real-Time Polymerase Chain Reaction - methods; real-time RT-PCR; Sensitivity and Specificity; Swine - virology; Typing; Viruses; Zoonoses
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v14020415

Avian influenza virus (AIV) variants emerge frequently, which challenges rapid diagnosis. Appropriate diagnosis reaching the sub- and pathotype level is the basis of combatting notifiable AIV infections. Real-time RT-PCR (RT-qPCR) has become a standard diagnostic tool. Here, a total of 24 arrayed RT-qPCRs is introduced for full subtyping of 16 hemagglutinin and nine neuraminidase subtypes of AIV. This array, designated Riems Influenza A Typing Array version 2 (RITA-2), represents an updated and economized version of the RITA-1 array previously published by Hoffmann et al. RITA-2 provides improved integration of assays (24 instead of 32 parallel reactions) and reduced assay volume (12.5 µL). The technique also adds RT-qPCRs to detect Newcastle Disease (NDV) and Infectious Bronchitis viruses (IBV). In addition, it maximizes inclusivity (all sequences within one subtype) and exclusivity (no intersubtypic cross-reactions) as shown in validation runs using a panel of 428 AIV reference isolates, 15 reference samples each of NDV and IBV, and 122 clinical samples. The open format of RITA-2 is particularly tailored to subtyping influenza A virus of avian hosts and Eurasian geographic origin. Decoupling and re-arranging selected RT-qPCRs to detect specific AIV variants causing epizootic outbreaks with a temporal and/or geographic restriction is possible.