Abstract
A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1–H13, H15, H16) and neuraminidase (N1–N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus.
Highlights
Influenza A virus circulating among humans and domestic and wild animals presents a significant pandemic threat due to the rapid evolution of strains with new antigenic properties [1]
Examples include the recent emergence of H1N1 swine influenza virus that proved pathogenic for humans, as well as H5N1 bird influenza virus that has the capacity to become pathogenic for domestic birds and humans
To reduce possible false positive hybridization results, oligonucleotide probes were tested for matches to other sub-types after introduction of one or two nucleotide changes
Summary
Influenza A virus circulating among humans and domestic and wild animals presents a significant pandemic threat due to the rapid evolution of strains with new antigenic properties [1]. We have explored the selection of probes based on amino acid sequences to create a universal microchip that could determine all sub-types of HA and NA of Influenza A virus. Nine sets of peptides that were 7 amino acids in length identifying each sub-type of NA were selected.
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