Universal primer set for amplification and sequencing of HA0 cleavage sites of all influenza A viruses.
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.
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
Astrid
A
Gall
Bernd
B
Hoffmann
Timm
T
Harder
Christian
C
Grund
Martin
M
Beer
D000482:Allantois / Q000821:virology; D000818:Animals; D000829:Animals, Domestic / Q000821:virology; D000835:Animals, Wild / Q000821:virology; D001717:Birds / Q000821:virology; D017931:DNA Primers / Q000235:genetics*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D009980:Influenza A virus / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011498:Protein Precursors; D012367:RNA, Viral