Abstract
Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA(0) is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA(0) cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA(0) cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay--followed by cycle sequencing--can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.
Publication types
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Comparative Study
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Allantois / virology
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Animals
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Animals, Domestic / virology
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Animals, Wild / virology
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Birds / virology
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DNA Primers / genetics*
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Hemagglutinin Glycoproteins, Influenza Virus / genetics*
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Influenza A virus / genetics*
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Influenza A virus / isolation & purification
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Molecular Sequence Data
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Protein Precursors / genetics
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RNA, Viral / genetics
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Reverse Transcriptase Polymerase Chain Reaction / methods*
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Sensitivity and Specificity
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Sequence Analysis, DNA
Substances
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DNA Primers
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Hemagglutinin Glycoproteins, Influenza Virus
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Protein Precursors
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RNA, Viral
Associated data
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GENBANK/AM922136
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GENBANK/AM922137
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GENBANK/AM922138
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GENBANK/AM922139
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GENBANK/AM922140
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