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Gewählte Publikation:

Publikationstyp: Zeitschriftenaufsatz
Dokumenttyp: Originalarbeit

Jahr: 2009

AutorInnen: Fereidouni, SR; Starick, E; Grund, C; Globig, A; Mettenleiter, TC; Beer, M; Harder, T

Titel: Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses.

Quelle: Vet Microbiol. 2009; 135(3-4):253-260

Autor/innen der Vetmeduni Vienna:

Fereidouni Sasan

Diese Publikation wurde nicht im Namen der Vetmeduni Vienna erstellt und ist deshalb ausschließlich der persönlichen Publikationsliste des/der Autors/Autorin zugeordnet!

Accurate identification of hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A viruses is an integral part of monitoring programs targeting avian influenza viruses (AIV). Use of highly sensitive molecular screening methods such as pan influenza-specific real-time RT-PCR (rRT-PCR) yields an increasing number of samples which are positive for AIV RNA but negative by virus isolation and, therefore, require molecular, instead of serological, subtyping. We developed specific RT-PCR assays for all known nine AIV NA subtypes. Validation using 43 reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity. On basis of serial tenfold dilution series of reference isolates a benchmark value of C(t) 32 in an M gene-specific rRT-PCR became evident below which all nine NA subtypes were readily detectable by the subtype-specific RT-PCRs. For subtypes N1, N2, N4 and N6 detection was extended to dilutions with C(t) values of up to 35. Diagnostic applicability of the whole set of conventional NA-specific RT-PCRs was evaluated by analysis of 119 different diagnostic samples from wild birds which proved to be positive for AIV by M gene-specific rRT-PCR. Diagnostic sensitivity and specificity was confirmed by sequencing NA amplicons from 41 field isolates generated from this set and by NA inhibition assays. A universal molecular HA/NA subtyping algorithm for rRT-PCR positive avian influenza virus monitoring samples is proposed which may complement classical serological subtyping of influenza A virus isolates.

Keywords Pubmed: Algorithms
Animals, Wild
Bird Diseases/virology
DNA Primers
DNA, Viral/chemistry
DNA, Viral/genetics
Influenza A Virus, H5N1 Subtype/genetics
Influenza A Virus, H5N1 Subtype/isolation & purification
Influenza A virus/genetics*
Influenza in Birds/genetics*
Neuraminidase/antagonists & inhibitors
Reverse Transcriptase Polymerase Chain Reaction/methods
Sensitivity and Specificity
United States

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