Abstract
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.
Highlights
Pathogenic avian influenza virus (HPAIV) is the major pathogen associated with substantial economic losses in poultry production
Due to the rapid evolutionary diversification of the Avian influenza virus (AIV) sequence cloud, affecting the HA segment, subtyping RT-qPCRs are under exceptionally high pressure to adapt to the fluctuations of such clouds
We show here that Riems Influenza A Typing Array version 2 (RITA-2) detects mixed infections in clinical samples even if these targets are present in grossly different concentrations (Figure 8)
Summary
Pathogenic avian influenza virus (HPAIV) is the major pathogen associated with substantial economic losses in poultry production. Zoonotic AIV strains, in addition, have caused multiple cases of human infections, sparking influenza pandemic concerns [1]. The influenza A virus genome consists of eight single-stranded RNA gene segments. Two segments encode the major envelope glycoproteins species of these viruses, hemagglutinin (HA) and neuraminidase (NA). These proteins have essential functions in defining host and tissue tropism and influence virulence. The HA, in particular, is a main target of the protective humoral immune response. Based on nucleotide sequence and protein antigenicity of the HA and NA surface glycoproteins, AIVs are classified into 16 different
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