Real-time reverse transcription PCR-based sequencing-independent pathotyping of Eurasian avian influenza A viruses of su
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Real-time reverse transcription PCR-based sequencing-independent pathotyping of Eurasian avian influenza A viruses of subtype H7 Annika Graaf, Martin Beer and Timm Harder*
Abstract Low pathogenic avian influenza viruses (LPAIV) of the subtypes H5 and H7 are known to give rise to highly pathogenic (HP) phenotypes by spontaneous insertional mutations which convert a monobasic trypsin-sensitive endoproteolytical cleavage site (CS) within the hemagglutinin (HA) protein into a polybasic subtilisin-sensitive one. Sporadic outbreaks of notifiable LPAIV H7 infections are continuously recorded in Europe and in Asia, and some lineages showed zoonotic transmission. De novo generation of HPAIV H7 from LPAIV precursors has been reported several times over the past decade. Rapid differentiation between LP and HP H7 virus strains is required as a prerequisite to emplace appropriate control measures. Here, reverse transcription real-time PCR assays (RT-qPCR) were developed and evaluated that allow LP and HP pathotype identification and distinction by probe-assisted detection of the HACS. These new RT-qPCRs allow a sensitive and highly specific pathotype identification of Eurasian subtype H7 AIV in allantoic fluids as well as in diagnostic field samples. RT-qPCR assisted pathotyping presents a rapid and sensitive alternative to pathotyping by animal inoculation or nucleotide sequencing. Keywords: Avian influenza, Hemagglutinin subtype H7, Pathotyping, Real-time RT-PCR, Diagnosis, Cleavage site
Background Avian influenza viruses (AIV) are members of the family Orthomyxoviridae, specified as influenza virus type-A. These viruses are further classified by the serologically defined subtypes of the predominant viral surface glycoproteins, the hemagglutinin (HA) and neuraminidase (NA) [1]. Their genome is composed of single-stranded, negative-sense RNA and comprises eight genome segments which encode at least ten proteins [2]. All 16 HA and nine NA AIV subtypes can be detected in populations of aquatic wild birds which form the natural reservoir of these viruses [3]. Based on their pathogenicity in chickens, two phenotypes of AIV are distinguished: highly pathogenic (HP) AIV and AIV of low pathogenicity (LPAIV). In nature, * Correspondence: [email protected] Friedrich Loeffler Institute, Institute of Diagnostic Virology, Südufer 10, Greifswald 17493, Germany
HP phenotypes have been restricted to viruses of subtypes H5 and H7. HPAIV arises from LPAI precursor viruses by spontaneous mutations leading to the insertion of basic amino acids into the cleavage site (CS) of the hemagglutinin protein (HA) which renders the HACS processible to subtilisin-like host proteases that are ubiquitous in all host tissues. Such viruses, therefore, gain competence for fatal systemic infections in avian hosts. LPAIV, in contrast, depends on local provision of trypsin-like proteases at the epithelial surfaces of the respiratory and/or gastrointestinal tracts and per se do not cause severe clinical signs [4]. All LPAIV and HPAIV
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